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K L Crossin

Publications and source records attributed to K L Crossin.

At least 19 recordsLinked to original sources
PubMed2000

N-CAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their differentiation to a neuronal phenotype.

Cell adhesion molecules (CAMs) play important roles during the development of the nervous system. On the basis of our previous observations that binding of the neural CAM (N-CAM) inhibits astrocyte proliferation and alters gene expression, we hypothesized that N-CAM may influence the balance between the proliferation and the differentiation of neural progenitor cells. Rat and mouse hippocampal progenitor cells were cultured and showed dependence on basic FGF for proliferation, immunoreactivity for nestin, the presence of limited numbers of differentiated cells, and the ability to generate glial cells and neurons under different culture conditions. Addition of soluble N-CAM reduced cell proliferation in a dose-dependent manner with no evidence of apoptosis. The inhibition of proliferation by N-CAM was accompanied by an induction of differentiation to the neuronal lineage, as indicated by a twofold increase in the percentage of microtubule-associated protein 2-positive cells even in the presence of mitogenic growth factors. Experiments using hippocampal cells from N-CAM knock-out mice indicated that N-CAM on the cell surface is not required for these effects, suggesting the existence of heterophilic signaling. These results support a role for N-CAM and N-CAM ligands in the inhibition of proliferation and the induction of neural differentiation of hippocampal neural progenitor cells.

Animals↗
PubMed2000

Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer.

Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the micro-calpain cleavage site from alpha-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.

Amino Acid Sequence↗
PubMed2000

Cellular signaling by neural cell adhesion molecules of the immunoglobulin superfamily.

Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult. In addition to their adhesive properties, binding of CAMs can affect intracellular signaling. Their ability to influence developmental events, including cell migration, proliferation, and differentiation can therefore result both from their adhesive as well as their signaling properties. This review focuses on the two CAMs for which the most information is known, the neural CAM, N-CAM, and L1. N-CAM was the first CAM to be characterized and, therefore, has been studied extensively. The binding of N-CAM to cells leads to a number of signaling events, some of which result in changes in gene expression. Interest in L1 derives from the fact that mutations in its gene lead to human genetic diseases including mental retardation. Much is known about modifications of the L1 cytoplasmic domain and its interaction with cytoskeletal molecules. The study of CAM signaling mechanisms has been assay-dependent rather than molecule-dependent, with particular emphasis on assays of neurite outgrowth and gene expression, an emphasis that is maintained throughout the review. The signals generated following CAM binding that lead to alterations in cell morphology and gene expression have been linked directly in only a few cases. We also review information on other CAMs, giving special consideration to those that are anchored in the membrane by a phospholipid anchor. These proteins, including a form of N-CAM, are presumed to be localized in lipid rafts, membrane substructures that include distinctive subsets of cytoplasmic signaling molecules such as members of the src-family of nonreceptor protein tyrosine kinases. In the end, these studies may reveal that what CAMs do after they bind cells together may have as profound consequences for the cells as the adhesive interactions themselves. This area will therefore remain a rich ground for future studies.

Adult↗
PubMed1999

NF-kappaB activity is induced by neural cell adhesion molecule binding to neurons and astrocytes.

The neural cell adhesion molecule, N-CAM, is expressed on the surface of astrocytes and neurons, and N-CAM homophilic binding has been shown to alter gene expression in both of these cell types. To determine mechanisms by which N-CAM regulates gene expression, we have analyzed DNA binding of and transcriptional activation by NF-kappaB after N-CAM binding to the cell surface. Addition of purified N-CAM, the recombinant third immunoglobulin domain of N-CAM, or N-CAM antibodies either to neonatal rat forebrain astrocytes or to cerebellar granule neurons increased NF-kappaB/DNA binding activity in nuclear extracts as measured by electrophoretic mobility shift assays. Analysis using supershifting antibodies indicated that, in both cell types, p50 and p65 but not p52, c-Rel, or Rel B were contained in the NF-kappaB-binding complex. NF-kappaB-mediated transcription was increased after N-CAM binding to astrocytes and neurons as demonstrated by the activation of two different luciferase reporter constructs containing NF-kappaB-binding sites. N-CAM binding also resulted in degradation of IkappaB-alpha protein followed by an increase in the levels of IkappaB-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappaB binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappaB, however, did not influence the previously reported ability of N-CAM to inhibit astrocyte proliferation. These observations together support the hypothesis that N-CAM binding activates multiple pathways leading to changes in gene expression in both astrocytes and neurons.

Animals↗
PubMed1998

Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.

Alkaline Phosphatase↗
PubMed1998

Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation.

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.

Animals↗
PubMed1998

Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM.

To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of beta-galactosidase under the control of the N-CAM promoter. Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of beta-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals. The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Treatment of the mice with an ampakine, an allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of beta-galactosidase in vivo as well as in tissue slices in vitro. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

Allosteric Regulation↗
PubMed1998

Domains of tenascin involved in glioma migration.

Tenascin (TN) is an extracellular matrix protein found in areas of cell migration during development and expressed at high levels in migratory tumor cells. TN was previously shown to support the attachment and migration of glioma cells in culture. To determine the domains responsible for glioma migration and attachment, we produced recombinant fusion proteins that collectively span the majority of the molecule including its epidermal growth factor-like repeats, fibronectin type III repeats and fibrinogen domain. These domains were tested for their ability to support migration of C6 glioma cells in an aggregate migration assay. A recombinant fusion protein including fibronectin type III (FNIII) repeats 2-6 (TNfn2-6) was the only fragment found to promote migration of C6 glioma cells at levels similar to that promoted by intact TN. Evaluation of smaller segments and individual FNIII repeats revealed that TNfn3 promoted migration and attachment of glioma cells and TNfn6 promoted migration but not attachment. While TNfn3 and TNfn6 promoted migration individually, the presence of both TNfn3 and TNfn6 was required for migration on segments of the FNIII region that included TNfn5. TNfn5 inhibited migration in a dose dependent manner when mixed with TNfn3 and also promoted strong attachment and spreading of C6 glioma cells. TNfn3 and TNfn6 promote cell migration and may function cooperatively to overcome the inhibitory activity of TNfn5. Additional cell attachment studies suggested that both beta1 integrins and heparin may differentially influence the attachment of glioma cells to TN fragments. Together, these findings show that C6 glioma cells integrate their response upon binding to at least three domains within TN.

Animals↗
PubMed1997

Developmental expression of two rat sialyltransferases that modify the neural cell adhesion molecule, N-CAM.

Polysialylation of the neural cell adhesion molecule (N-CAM) reduces the efficacy of N-CAM-mediated homophilic binding and is regulated both during development and in regions undergoing neurogenesis or remodeling in the adult. Hamster PST-1 (PST) and rat STX are two related sialytransferases that catalyze the polysialylation of N-CAM. We have isolated a cDNA clone for the rat homologue of PST and compared its amino acid and nucleotide sequence to that of rat STX. This analysis revealed regions of high sequence similarity corresponding to the enzymatic domains of the two molecules. Other regions of lower similarity were used to generate specific probes for in situ hybridization. The distribution of PST and STX mRNAs, polysialic acid, and N-CAM were analyzed at three developmental stages. PST and STX mRNAs were expressed abundantly throughout the nervous system at embryonic day 15 and postnatal day 4 and were coexpressed in most tissues examined. In the adult brain, STX expression was reduced relative to PST and expression of both mRNAs was restricted to subsets of cells in areas undergoing constant synaptic rearrangement including hippocampus and olfactory system. The results suggest that both PST and STX participate in the polysialylation of N-CAM in vivo and that their expression levels are dynamically controlled during development and regeneration.

Amino Acid Sequence↗
PubMed1997

Glucocorticoid receptor pathways are involved in the inhibition of astrocyte proliferation.

In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes both in vitro and in vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAM in vitro. While the mRNA levels for N-CAM decreased after such treatment, the levels of mRNAs for glutamine synthetase and calreticulin increased. Since glutamine synthetase and calreticulin are known to be involved in glucocorticoid receptor pathways, we tested a number of steroids for their effects on astrocyte proliferation. Dexamethasone, corticosterone, and aldosterone were all found to inhibit rat cortical astrocyte proliferation in culture in a dose-dependent manner. RU-486, a potent glucocorticoid antagonist, reversed the inhibitory effects of dexamethasone. These observations prompted the hypothesis that the inhibition of proliferation by N-CAM might be mediated through the glucocorticoid receptor pathway. Consistent with this hypothesis, the inhibition of astrocyte proliferation by N-CAM was reversed in part by a number of glucocorticoid antagonists, including RU-486, dehydroepiandrosterone, and progesterone. In transfection experiments with cultured astrocytes, N-CAM treatment increased the expression of a luciferase reporter gene under the control of a minimal promoter linked to a glucocorticoid response element. The enhanced activity of this construct stimulated by N-CAM was abolished in the presence of RU-486. The combined data suggest that astrocyte proliferation is in part regulated by alterations in glucocorticoid receptor pathways.

Aldosterone↗
PubMed1996

Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.

To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

Amino Acid Sequence↗
PubMed1996

Migration of luteinizing hormone-releasing hormone (LHRH) neurons in early human embryos.

Luteinizing hormone-releasing hormone (LHRH) neurons originate in the epithelium of the medial olfactory pit and migrate from the nose into the forebrain along nerve fibers rich in neural cell adhesion molecule (N-CAM). The present study examined the ontogenesis of LHRH neurons in early human embryos and found a similar pattern of development of these cells. Luteinizing hormone-releasing hormone immunoreactivity was detected in the epithelium of the medial olfactory pit and in cells associated with the terminal-vomeronasal nerves at 42 (but not 28-32) days of gestation. The migration route of these cells was examined with antibodies to N-CAM and antibodies to polysialic acid (PSA-N-CAM), which is present on N-CAM at certain stages of development. Neural cell adhesion molecule immunoreactivity was present in a population of cells in the olfactory placode of the earliest embryos examined (28-32 days) and later (42 and 46 days) throughout the migration route. The PSA-N-CAM immunoreactivity was not detected until 42 days and was present in a more limited distribution in nerve fibers streaming from the olfactory placode and along the caudal part of the migration route below the forebrain. Previous studies have indicated that the highly sialated form of N-CAM is less adhesive. The PSA-N-CAM may therefore facilitate the migration of these cells by lessening the adhesion between the fascicles that make up the migration route, expediting the passage of cords of LHRH cells between the nerve fibers as these cells move toward the brain.

Antibodies, Monoclonal↗
PubMed1995

Neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation after injury to different regions of the adult rat brain.

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately 20%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic fibroblast growth factor, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.

Amino Acid Sequence↗
PubMed1995

Protein H--a bacterial surface protein with affinity for both immunoglobulin and fibronectin type III domains.

Several bacterial species express surface proteins with affinity for the constant region (Fc) of immunoglobulin (Ig) G. The biological consequences of the interaction with IgG are poorly understood but it has been demonstrated that genes encoding different IgG Fc-binding proteins have undergone convergent evolution, suggesting that these surface molecules are connected with essential microbial functions. One of the molecules, protein H, is present in some strains of Streptococcus pyogenes, the most significant streptococcal species in clinical medicine. In contrast to other Ig-binding bacterial proteins tested, protein H was found to interact also with the neural cell adhesion molecule (N-CAM), a eukaryotic cell surface glycoprotein mediating homo- and heterophilic cell-cell interactions. The affinity for the interaction between protein H and N-CAM was 1.6 x 10(8)/M and the binding site on protein H was mapped to the NH2-terminal 80 amino acid residues. N-CAM and IgG are both members of the Ig superfamily and analogous to N-CAM, IgG binds to the NH2-terminal part of protein H. However, the binding sites for the two proteins were found to be separate, an unexpected result which was explained by the observation that the fibronectin type III (FNIII) domains and not the Ig-like domains of N-CAM are responsible for the interaction with protein H. Thus, the binding of N-CAM to protein H was blocked with fibronectin but not with IgG. Moreover, apart from fibronectin itself and N-CAM, fragments of fibronectin and the matrix protein cytotactin/tenascin containing FNIII domains also showed affinity for protein H.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

The neural cell adhesion molecule (N-CAM) inhibits proliferation in primary cultures of rat astrocytes.

Cell proliferation is a key primary process during neural development and also plays an important role in the regenerative response of neural tissue to injury. It has been reported that glial cell proliferation is, at least in part, controlled by a neuronal signal, possibly involving cell surface molecules. We report here that the addition of purified rat neural cell adhesion molecule (N-CAM) to primary cultures of rat forebrain astrocytes inhibits their proliferation. This inhibitory effect can be elicited in cultures grown in chemically defined serum-free medium or in medium that had been supplemented with growth factors. Polyclonal antibodies to N-CAM or their Fab' fragments elicited a similar inhibitory effect. The magnitude of the inhibitory effect of N-CAM was dependent on cell density: it was maximal at low cell densities and weakened progressively as cells approached confluency. Synthetic peptides with sequences identical to a putative homophilic binding region of N-CAM mimicked the effect of purified N-CAM, while peptides of the same length and amino acid composition but with a randomized sequence did not. The addition of N-CAM antisense oligonucleotides to primary astrocyte cultures for 48 h resulted in reduced levels of N-CAM expression. After N-CAM levels on astrocytes were diminished by this treatment, the antiproliferative effect of N-CAM added to the medium was significantly reduced. The combined results suggest that N-CAM homophilic binding may be involved in the control of glial cell proliferation.

Animals↗
PubMed1995

EGF receptor-mediated signals are differentially modulated by concanavalin A.

NIH 3T3 cells expressing high levels of the human epidermal growth factor (EGF) receptor were used to examine the effects of the lectin concanavalin A (Con A) on EGF-mediated signaling events. Proliferation of NIH 3T3 cells expressing high levels of the human EGF receptor was inhibited in a dose-dependent manner by Con A. At the same time, Con A also inhibited both dimerization and tyrosine phosphorylation of the EGF receptor. Tyrosine phosphorylation of the enzyme phospholipase C-gamma, a substrate of the phosphorylated EGF receptor kinase, was also inhibited. In contrast, EGF-stimulated changes in pH, calcium, and levels of inositol phosphates were unaffected by the presence of Con A. These results indicate that certain signals (changes in the levels of intracellular calcium, pH, and inositol phosphates) mediated by EGF binding to its receptor still occur when receptor dimerization and phosphorylation are dramatically decreased, suggesting that multiple independent signals are transmitted by the binding of EGF to its receptor.

Cell Division↗
PubMed1995

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM.

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Amino Acid Sequence↗