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 have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca 2؉ -independent immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3␣ (biggest), -3␤ (middle), and -3␥ (smallest). Like nectin-1 and -2, nectin-3␣ consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3␣ formed a cis-homo-dimer and showed Ca 2؉ -independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3␣ furthermore showed trans-hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-heterointeraction with nectin-2. The affinity of trans-heterointeraction of nectin-3␣ with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3␣. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3␣ was colocalized with nectin-2 at cadherinbased AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs.

We have recently found a novel functional unit of cellcell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2␣, a truncated form of nectin-2␣ incapable of interacting with afadin (nectin-2␣-⌬C), or a point-mutated form of nectin-2␣ capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2␣. We found that the interaction of nectin-2␣ with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2␣-⌬C showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2␣ with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2␣ was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.We have recently found a novel functional unit of cell-cell adhesion at cadherin-based AJs 1 (1, 2). This adhesion unit consists of at least nectin and afadin. Nectin is a Ca 2ϩ -independent homophilic CAM, which belongs to the Ig superfamily (2-7). Human nectin is identical to the poliovirus receptorrelated protein (3-6) and has recently been identified to be the ␣-herpesvirus entry mediator (8, 9). Nectin constitutes a family consisting of at least nectin-1 and -2 (2-7). Nectin-2 has two splicing variants, nectin-2␣ and -2␦. Each member of the nectin family consists of extracellular three Ig-like domains, a single transmembrane region, and a single cytoplasmic region. The cytoplasmic region has a C-terminal conserved motif of four amino acid residues, which interacts with the PDZ domain of afadin, an actin filament-binding protein, and is linked to the actin cytoskeleton through afadin (1, 2). Afadin has two splicing variants, l-and s-afadins (1). Human s-afadin is identical to AF-6, the gene of which is originally found to be fused to the ALL-1 gene in acute leukemia (10). The interaction of nectin-1 with afadin is essential for its recruitment to cadherin-based AJs (2).Cadherin is a Ca 2ϩ -dependent homophilic CAM that plays a fundamental role in cell-cell adhesion (for review, see Refs. 11-16). Cadherin typically consists of extracellular five tandemly repeated domains, EC1-EC5, a single transmembrane region, and a cytoplasmic region (for review, see Refs. 15-18). The distal portion of the cytoplasmic region interacts with catenins, including ␣-, ␤-, and ␥-catenins, which connect cadherin to the actin cytoskeleton. The juxtamembrane portion interacts with p120ctn (19,20). Cadherin forms a ci...

We recently found a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs), consisting at least of nectin, a Ca 2؉ -independent homophilic immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin is associated with cadherin through afadin and ␣-catenin. The cadherin-catenin system increases the concentration of nectin at AJs in an afadin-dependent manner. Nectin constitutes a family consisting of three members: nectin-1, -2, and -3. Nectin-1 serves as an entry and cell-cell spread mediator of herpes simplex virus type 1 (HSV-1). We studied here a role of the interaction of nectin-1␣ with afadin in entry and/or cell-cell spread of HSV-1. By the use of cadherin-deficient L cells overexpressing the full length of nectin-1␣ capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1␣ incapable of interacting with afadin, we found that the interaction of nectin-1␣ with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1␣ of glycoprotein D, a viral component mediating entry of HSV-1 into host cells. Furthermore, the cadherin-catenin system increased the efficiency of cell-cell spread of HSV-1, although it also increased the efficiency of entry of HSV-1. It is likely that efficient cell-cell spread of HSV-1 is caused by afadin-dependent concentrated localization of nectin-1␣ at cadherin-based AJs.Herpes simplex viruses (HSVs) are members of the neurotropic subfamily (alphaherpesviruses) of the herpesvirus family. Infection with HSV type 1 (HSV-1) is prevalent. HSV-1 infects cells through initial attachment to the plasma membrane and subsequent fusion of the viral envelope with the plasma membrane or through contiguous cell-cell spread. The entry pathway of HSV-1 is divided into three major processes: binding, fusion, and capsid penetration. These processes require several viral envelope glycoproteins (49-51). The initial attachment is mediated through glycoprotein C (gC) and/or gB to cell surface heparan sulfate proteoglycans (17,18,45), but this attachment is not sufficient for virus penetration (3,11,22,27). The fusion of the viral envelope with the plasma membrane requires gD, gB, gH, and gL (49-51). These four viral glycoproteins also participate in cell-cell spread (3,11,20,28,36,40). Cell-cell spread furthermore requires gE and gI (1, 8-10), but these glycoproteins are not required for entry (8). Thus, entry and cell-cell spread of HSV-1 share similar processes, but these pathways also differ in some significant aspects.Recently, expression cloning has led to the identification and isolation of HSV entry mediators (5,12,33,46,51). The human receptors identified include a lymphotoxin receptor (31), designated HVEM (33, 65) or HSV entry mediator A (HveA), which belongs to the tumor necrosis factor receptor family; two members of the immunoglobulin (Ig) superfamily (5,12,48,62); and 3-O-sulfated heparan ...

We purified from rat brain a novel F-actin-binding protein with a M r of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated M r of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.