Nectins are Ca2؉ -independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins. Four nectins have been identified. Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca 2؉ -independent homophilic cell-cell adhesion activity. It furthermore showed Ca 2؉ -independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3. Necl-2 was widely expressed in rat tissues examined. Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2.

Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin-and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins.In multicellular organisms, cell adhesion and migration are critical for many events, including tissue patterning, morphogenesis, and maintenance of normal tissues (1-3). They also play roles in malignant transformation of cells (4). Adhesion and migration of non-transformed normal cells are dynamic and well regulated (2). Cells disrupt cell-cell adhesion and start to migrate in response to extracellular cues, such as growth factors, cytokines, and extracellular matrix molecules (4). When migrating cells contact other cells, they stop migration and proliferation and adhere to each other to become confluent (5, 6). This phenomenon is known for a long time as contact inhibition of cell movement and proliferation. Transformation of cells causes disruption of cell-cell adhesion, increase of cell motility, and loss of contact inhibition of cell movement and proliferation, eventually leading the transformed cells to invasion into surrounding tissues and metastasis to other organs (4, 7). However, molecular mechanisms underlying these physiological or pathological processes are not fully understood.Cell-cell adherens junctions (AJs) 1 play major roles in cellcell adhesion in fibroblasts and epithelial cells (1, 2). Cadherins are key Ca 2ϩ -dependent cell-cell adhesion molecules at AJs (1, 2). Cadherins are associated with the actin cytoskeleton through peripheral membrane proteins, including ␣-and ␤-catenins, in fibroblasts and epithelial cells (1). This association strengthens the cell-cell adhesion activity of cadherins (1). Nectins and afadin constitute another cell-cell adhesion unit that localizes at cell-cell AJs and regulates organization of AJs in cooperation with cadherins in fibroblasts and epithelial cells (8). Nectins are Ca 2ϩ -independent Ig-like cell-cell adhesion molecules. Afadin is a nectin-and actin filame...

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules and comprise a family of four members. At the mossy fiber terminals of hippocampus, nectin-1 and nectin-3 localize at the presynaptic and postsynaptic sides of synaptic junctions, respectively, and their trans-interactions play a role in formation of synapses in cooperation with N-cadherin. Nectins are associated with the actin cytoskeleton through afadin, a nectin- and actin-filament-binding protein. Five nectin-like molecules (Necls) which have domain structures similar to those of nectins have been identified and here we characterize Necl-1/TSLL1/SynCAM3, from now on referred to as Necl-1. Tissue distribution analysis showed that Necl-1 was specifically expressed in the neural tissue. Immunofluorescence and immunoelectron microscopy revealed that Necl-1 localized at the contact sites among axons, their terminals, and glia cell processes that cooperatively formed synapses, axon bundles and myelinated axons. Necl-1 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 from now on referred to as Necl-2, nectin-1 and nectin-3, but not with Necl-5 or nectin-2. The C-terminal cytoplasmic region of Necl-1 did not bind afadin but bound membrane-associated guanylate kinase subfamily members that contain the L27 domain, including Dlg3, Pals2 and CASK. These results indicate that Necl-1 is a neural-tissue-specific Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which potentially has membrane-associated guanylate kinase subfamily member-binding activity and localizes at the non-junctional cell-cell contact sites.

Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor–induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell–cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell–cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell–cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca 2؉ -independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum-and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin ␣ V ␤ 3 at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin ␣ V ␤ 3 or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin ␣ V ␤ 3 in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum-and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.In multicellular organisms, cell migration is essential for normal development and responses to tissue damages and infection throughout life (1, 2). Cell migration is also observed in many diseases, such as cancer and atherosclerosis (3, 4). Cells migrate as individuals or as groups; leukocytes, lymphocytes, and fibroblasts migrate as individuals, whereas epithelial and endothelial cells migrate as groups. Cell migration is divided into at least four mechanistically separate steps: extension of protrusions, formation of new cell-matrix adhesions, contraction of cell body, and tail detachment (1, 5). Cell migration is normally directed and controlled by extracellular cues, such as growth factors, cytokines, and extracellular matrix molecules. These cues stimulate cell surface receptors to initiate intracellular signaling through second messengers, protein kinases, protein phosphatases, and heterotrimeric large and monomeric small G proteins to regulate the multiple steps. When migrating cells contact other cells, they stop migration and proliferation (6, 7). This phenomenon is known for a long time as contact inhibition of cell movement and proliferation. Transformation of cells causes disruption of cell-cell adhesion, increase of cell motility, and loss of contact inhibition of cell movement and proliferation, eventually leading transformed cells to invasion into surrounding t...