Characterization of the biosynthesis,membrane association and function of the cell adhesion molecule L1

The L1 cell adhesion molecule is involved in cell migration and cell-cell adhesion in the brain. In this report we describe a purification procedure which allowed simultaneous isolation of L1 and the neural cell adhesion molecule. Furthermore, we studied L1 biosynthesis, post-translational modifications and function. L1 was synthesized as a polypeptide with relative molecular weight 200,000 in monolayer cultures of fetal rat neurons and in explant cultures of rat forebrain. The L1 polypeptide was co-translationally inserted into the membranes of the endoplasmic reticulum as an integral membrane protein. Both sulfation and phosphorylation of L1 was observed. L1 polypeptides with lower relative molecular weight which were present on the plasma membrane were probably derived from the 200,000 mol. wt polypeptide. The function of L1 was investigated and an L1 involvement in neurite fasciculation was demonstrated.     

Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.     

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.     

Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin

The role of calcium-dependent adhesion molecules in the migration of nerve growth cones onto the top of Schwann cells was probed by examination of sensory growth cone-Schwann cell interactions in medium containing either 1.0 mM Ca2+ or 0.1 mM Ca2+. In the presence of 1.0 mM Ca2+ growth cones rapidly migrated onto Schwann cells, spread, and remained for extended periods. However, in 0.1 mM Ca2+ growth cones still made frequent contacts with Schwann cells, but migration onto the upper cell surface was much reduced. This contrast in growth cone-Schwann cell interactions could be switched rapidly by changing the Ca2+ concentration of the culture medium. Growth cones of retinal neurons showed similar calcium-dependence in their migration onto Schwann cells. Antibodies to the calcium-dependent adhesion molecule, N-cadherin, also blocked growth cone migration onto Schwann cells, but antibodies to another neuronal adhesion molecule, L1, had no effect on growth cone-Schwann cell interactions. Immunocytochemical staining for N-cadherin and L1 indicated that growth cones and Schwann cells have N-cadherin on their surfaces, while L1 is present only on axons and growth cones. These results provide two kinds of evidence that N-cadherin is important in the initial interactions of growth cones and Schwann cells.     

The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.     

Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.     

Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell-cell adhesion molecule, platelet-endothelial cell adhesion molecule-1, (PECAM-1), a member of the lg superfamily, in the modulation of cell migration and cell-cell adhesion. PECAM-1 is a 120-130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell-cell contact. Since endothelial cells express PECAM-1 constitutively, we studied the effects of PECAM-1 on cell-cell adhesion and migration in a null-cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM-1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM-1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM-1 transfectants showed peripheral staining at sites of cell-cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full-length PECAM-1 transfectants migrated more slowly than control cells. Thus, PECAM-1 transfected into a null cell appears to localize to sites of cell-cell contact, promote cell-cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell-cell adhesion molecule in the process of endothelial cell migration.     

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.     

Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

Endocytosis of beta1 integrins is an early event in migration promoted by the cell adhesion molecule L1

Directional cell motility is a complex process requiring orchestration of signals from diverse cell adhesion receptors for proper organization of neuronal groups in the brain. The L1 cell adhesion molecule potentiates integrin-dependent migration of neuronal cells and stimulates integrin endocytosis but its mechanism of action is unclear. The hypothesis was investigated that L1 stimulates cell motility by modulating surface levels of integrins through intracellular trafficking using a model cell system. Antibody-induced clustering of L1, which mimics ligand binding, induced formation of cell surface complexes of L1 and beta1 integrins in L1-expressing HEK293 cells. L1 formed cell surface complexes with integrin beta1 and alpha3 subunits but not with integrin alpha1. Following cell surface clustering, beta1 integrins and L1 became rapidly internalized into Rab5+ early endosomes. Internalization of L1 and beta1 integrins was prevented by treatment with monodansyl cadaverine (MDC), an inhibitor of clathrin-dependent endocytosis, and by deletion of the AP2/clathrin binding motif (RSLE) from the L1 cytoplasmic domain. MDC treatment coordinately inhibited L1-potentiated haptotactic migration of HEK293 cells to fibronectin in Transwell assays. These results suggested that downregulation of adhesive complexes of L1 and beta1 integrin at the plasma membrane by clathrin-mediated endocytosis is a potential mechanism for enhancing cell motility.     

The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system.

Cortical nonpyramidal cells, the GABA-containing interneurons, originate mostly in the medial ganglionic eminence of the ventral telencephalon and follow tangential migratory routes to reach the dorsal telencephalon. Although several genes that play a role in this migration have been identified, the underlying cellular and molecular cues are not fully understood. We provide evidence that the neural cell adhesion molecule TAG-1 mediates the migration of cortical interneurons. We show that the migration of these neurons occurs along the TAG-1-expressing axons of the developing corticofugal system. The spatial and temporal pattern of expression of TAG-1 on corticofugal fibers coincides with the order of appearance of GABAergic cells in the developing cortex. Blocking the function of TAG-1, but not of L1, another adhesion molecule and binding partner of TAG-1, results in a marked reduction of GABAergic neurons in the cortex. These observations reveal a mechanism by which the adhesion molecule TAG-1, known to be involved in axonal pathfinding, also takes part in neuronal migration.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca²⁺ signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.     

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.     

Glycoproteins and lectins in cell adhesion and cell recognition processes

Summary The discovery of endogenous lectins having specific and high affinity for the carbohydrate portions of glycoproteins has opened up new directions in the field of cell adhesion and cell recognition. Two endogenous lectins, termed as CSL and R1, initially isolated from the rat cerebellum and having a wide distribution in mammalian tissues, have been shown to participate in essential mechanisms of cell adhesion. The membrane-bound lectin R1 seems to be involved in transient recognition between neuronal cells, followed by elimination of the glycoprotein ligands at the surface of the recognized cell. In contrast, CSL is a molecule involved in adhesion between various normal or transformed cells since it participates in the formation of tight junctions. The glycoprotein ligands recognized with higher affinity by these two lectins seem to possess a special structure which defines a sub-class of oncofetal HNK-1 glycans. The over-expression of the glycoprotein ligands of these lectins in most transformed cells provides new tools for understanding the underlying mechanism of malignant transformation as well as the generation of signals through cell adhesion.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

The roles of cell adhesion molecules in tumor suppression and cell migration

In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.     

Separation force measurements reveal different types of modulation of E-cadherin-based adhesion by nectin-1 and -3

Nectins are Ca2+-independent cell adhesion molecules found at cadherin-based adherens junctions. We used a dual pipette assay that measures the forces required to separate cell doublets to determine how nectins affect the formation and strength of cell-cell adhesion. Less force was required to separate doublets of L cells expressing nectin-1 or nectin-3 than to separate doublets of E-cadherin-expressing cells. Heterodimers formed between cells expressing nectin-1 or nectin-3 adhered more strongly than homodimers. Nectin-3 that does not trans-interact with nectin-1 inhibited E-cadherin-mediated adhesion. However, the extracellular fragment of nectin-1 did not have an agonistic effect on E-cadherin-dependent cell adhesion when it trans-interacted with nectin-3, expressed at high levels in cells. In contrast, the extracellular fragment of nectin-3 had a significant agonistic effect on cadherin-based adhesion when it interacted with endogenous nectin-1, expressed at low levels in cells. Our results indicate that E-cadherin is the key molecule involved in cell adhesion and that the regulation of E-cadherin-based adhesion involving cellular nectin-1 trans-interacting with nectin-3 is qualitatively different from that involving cellular nectin-3 trans-interacting with nectin-1 and depends on the nectin levels expressed by cells.