We have isolated a novel actin filament–binding protein, named afadin, localized at cadherin-based cell–cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament–binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor–related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell–cell AJs in various tissues and cell lines. In E-cadherin–expressing EL cells, PRR was recruited to cadherin-based cell–cell AJs through interaction with afadin. PRR showed Ca2+-independent cell–cell adhesion activity. These results indicate that PRR is a cell–cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell–cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word “necto” meaning “to connect”).

We recently isolated a novel actin filament (F-actin)–binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell–cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin–binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin–binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells, we attempted here to identify an l-afadin–binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin–binding protein localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin– and vinculin-binding protein localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells.

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.

We purified from rat brain a novel actin filament (F-actin)–binding protein of ∼180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue–specific F-actin– binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin–binding domain at the NH2-terminal region, one PSD-95, DlgA, ZO-1–like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin–cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.

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...

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

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...

In a preceding paper, we reported a novel actin filament (F-actin)-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation. We purified from rat brain another F-actin-binding protein, which had a domain organization similar to that of neurabin but was ubiquitously expressed, and named it neurabin-II. The original neurabin, renamed neurabin-I, had 1095 amino acids and a calculated M r of 122,729, whereas neurabin-II had 817 amino acids and a calculated M r of 89,642. Both neurabin-I and -II had one F-actin-binding domain at the N-terminal region, one PDZ domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiledcoil structures at the C-terminal region. Both neurabin-I and -II bound along the sides of F-actin and showed F-actin-cross-linking activity. The subcellular distribution analysis indicated that neurabin-II was enriched at the postsynaptic density fraction in rat brain and the adherens junction fraction in rat liver. Immunofluorescence microscopic analysis revealed that neurabin-II was highly concentrated at the synapse in primary cultured rat hippocampal neurons and at the cadherinbased cell-cell adhesion sites in Madin-Darby canine kidney cells. Neurabin-II turned out to be the same as a recently reported protein phosphatase 1-binding protein named spinophilin. These results suggest that neurabin-II/spinophilin plays an important role in linking the actin cytoskeleton to the plasma membrane.Specialized membrane domains formed with transmembrane proteins, such as cell adhesion molecules, receptors, and channels, are often associated with the actin cytoskeleton (for reviews, see Refs. 1-4). The linkage between the actin cytoskeleton and the plasma membrane plays crucial roles in various cellular events, such as cell adhesion, cell motility, and cell shape determination, and the proteins linking the actin cytoskeleton to the transmembrane proteins have been identified (1-4). However, the molecular basis of this linkage is not fully understood.In a preceding paper, we purified from rat brain a novel F-actin-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation (5). Neurabin had one F-actin-binding domain, one PDZ domain, and four domains predicted to form coiled-coil structures. The PDZ domain is found in many proteins, some of which are localized at cell-cell junctions (for review, see Ref. 6), such as PSD-95/SAP90 at synaptic junction, Dlg at septate junction, and ZO-1 and ZO-2 at tight junction. The PDZ domain binds to the unique C-terminal motifs of target proteins found in many transmembrane proteins, such as N-methyl-D-aspartate receptors and Shaker-type K ϩ channels (6). Neurabin is likely to serve as a linker between the actin cytoskeleton and a transmembrane protein(s) at synapse, although we have not yet identified its interacting transmembrane protein.During the purification of neurabin, we detected another F-...