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”).

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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.

Adipose tissue secretes adipokines that mediate insulin resistance, a characteristic feature of obesity and type 2 diabetes. By differential proteome analysis of cellular models of insulin resistance, we identified progranulin (PGRN) as an adipokine induced by TNF-α and dexamethasone. PGRN in blood and adipose tissues was markedly increased in obese mouse models and was normalized with treatment of pioglitazone, an insulin-sensitizing agent. Ablation of PGRN (Grn(-/-)) prevented mice from high fat diet (HFD)-induced insulin resistance, adipocyte hypertrophy, and obesity. Grn deficiency blocked elevation of IL-6, an inflammatory cytokine, induced by HFD in blood and adipose tissues. Insulin resistance induced by chronic administration of PGRN was suppressed by neutralizing IL-6 in vivo. Thus, PGRN is a key adipokine that mediates HFD-induced insulin resistance and obesity through production of IL-6 in adipose tissue, and may be a promising therapeutic target for obesity.

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.

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...

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.

Mucopolysaccharidosis II (MPS II) is an X-linked recessive lysosomal storage disease caused by mutations in the iduronate-2-sulfatase (IDS) gene. Since IDS catalyzes the degradation of glycosaminoglycans (GAGs), deficiency in this enzyme leads to accumulation of GAGs in most cells in all tissues and organs, resulting in severe somatic and neurological disorders. Although enzyme replacement therapy with human IDS (hIDS) has been used for the treatment of MPS II, this therapy is not effective for defects in the CNS mainly because the enzyme cannot cross the blood-brain barrier (BBB). Here, we developed a BBB-penetrating fusion protein, JR-141, which consists of an anti-human transferrin receptor (hTfR) antibody and intact hIDS. The TfR-mediated incorporation of JR-141 was confirmed by using human fibroblasts in vitro. When administrated intravenously to hTfR knockin mice or monkeys, JR-141, but not naked hIDS, was detected in the brain. In addition, the intravenous administration of JR-141 reduced the accumulation of GAGs both in the peripheral tissues and in the brain of hTfR knockin mice lacking Ids, an animal model of MPS II. These data provide a proof of concept for the translation of JR-141 to clinical study for the treatment of patients with MPS II with CNS disorders.