Quantitative analysis of intercellular adhesive specificity in freshly explanted and cultured cells.

257

340

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.

Section: Methodsmentioning

confidence: 99%

Nectin-3, a New Member of Immunoglobulin-like Cell Adhesion Molecules That Shows Homophilic and Heterophilic Cell-Cell Adhesion Activities

Satoh-Horikawa

Nakanishi

Takahashi

et al. 2000

257

340

“…The labeled nectin-2␣-L cells were mixed with an equal number of pm-nectin-2␣-L cells or nectin-2␣-⌬C-L cells to give a final concentration of 1 ϫ 10 6 cells/ml and rotated at 37°C for 30 min. After the addition of 2% glutaraldehyde, aggregates were subjected to phase and fluorescence microscopy, and the cell composition (labeled or unlabeled cells) of four-cell aggregates was determined as described (39).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Nectin with Afadin Is Necessary for Its Clustering at Cell-Cell Contact Sites but Not for Itscis Dimerization or trans Interaction

Miyahara¹,

Nakanishi²,

Takahashi³

et al. 2000

135

180

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...

“…The number of fluorescent cells in each aggregate of a given size was counted. Quantitative analysis of the aggregating cell populations was performed as described by Sieber and Roseman (28).…”

Section: Construction Of Cytoplasmic Domain Mutants Of Hupecam-1 (Seementioning

confidence: 99%

Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Homophilic Adhesion Is Mediated by Immunoglobulin-like Domains 1 and 2 and Depends on the Cytoplasmic Domain and the Level of Surface Expression

Sun

Yan

et al. 1996

133

122

PECAM-1/CD31 is vascular cell adhesion and signaling molecule of the Ig superfamily that plays a role in neutrophil recruitment at inflammatory sites and may be involved the release of leukocytes from the bone marrow and in cardiovascular development. The interactions of PECAM-1 with its ligands are complex in that it is able to bind both with itself (homophilic adhesion) or with non-PECAM-1 ligands (heterophilic adhesion). Although the factors that regulate ligand binding are not fully understood, these interactions are regulated in part by its large cytoplasmic domain, a region of 118 amino acids encoded by 8 exons of its gene (exons 9 -16). The purpose of this work was to better define the mechanisms of PECAM-1-dependent homophilic adhesion by analyzing the binding interactions of L-cells expressing full-length and selectively mutated forms of human, murine, and human/murine chimeric PECAM-1 molecules in an established aggregation assay. These studies demonstrate that 1) the minimal length of the cytoplasmic domain required for cellular aggregation is represented within the sequences encoded by exons 9 and 10, 2) removal or addition of the sequences encoded by exon 14 from the cytoplasmic domain can determine whether the mechanism of aggregation is a heterophilic calciumdependent process or a homophilic calcium-independent process, 3) high levels of surface expression of PE-CAM-1 on the cell surface change the mechanism of aggregation from heterophilic to homophilic, and 4) PE-CAM-1-dependent homophilic binding appears to involve the direct interaction of only the first two extracellular Ig-like domains. These data suggest that PECAM-1-ligand interactions can be regulated through multiple pathways including alterations of the cytoplasmic domain and the level of surface expression.