Involvement of nectin in inactivation of integrin alpha(v)beta(3) after the establishment of cell-cell adhesion

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.     

Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions

Nectins, Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, induce the activation of Cdc42 and Rac small G proteins, enhancing the formation of cadherin-based adherens junctions (AJs) and claudin-based tight junctions. Nectins recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then activates Cdc42 through FRG, a Cdc42-GDP/GTP exchange factor. We showed here that Rap1 small G protein was involved in the nectin-induced activation of Cdc42 and formation of AJs. Rap1 was recruited to the nectin-based cell-cell contact sites and locally activated through the c-Src-Crk-C3G signaling there. The activation of either c-Src or Rap1 alone was insufficient for and the activation of both molecules was essential for the activation of FRG. The activation of Rap1 was not necessary for the c-Src-mediated phosphorylation or recruitment of FRG. The inhibition of the Crk, C3G, or Rap1 signaling reduced the formation of AJs. These results indicate that Rap1 is activated by nectins through the c-Src-Crk-C3G signaling and involved in the nectin-induced, c-Src- and FRG-mediated activation of Cdc42 and formation of AJs.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Nectins, Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, initiate cell-cell adhesion by their trans interactions and recruit cadherins to cooperatively form adherens junctions (AJs). In addition, the trans interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which increases the velocity of the formation of AJs. We examined here how nectins induce the activation of Cdc42 in MDCK epithelial cells and L fibroblasts. Nectins recruited and activated c-Src at the nectin-based cell-cell adhesion sites. FRG, a GDP/GTP exchange factor specific for Cdc42, was then recruited there, tyrosine phosphorylated by c-Src, and activated, causing an increase in the GTP-bound active form of Cdc42. Inhibition of the nectin-induced activation of c-Src suppressed the nectin-induced activation of FRG and Cdc42. Inhibition of the nectin-induced activation of FRG or depletion of FRG by RNA interference suppressed the nectin-induced activation of Cdc42. These results indicate that nectins induce the activation of Cdc42 through c-Src and FRG locally at the nectin-based cell-cell adhesion sites.     

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.     

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.     

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.     

Role of nectin in formation of E-cadherin-based adherens junctions in keratinocytes: analysis with the N-cadherin dominant negative mutant

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin-based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin-based AJs in keratinocytes.     

Roles and modes of action of nectins in cell-cell adhesion

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell-cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell-cell junctions and cell-cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell-cell adhesion and recruit E-cadherin to the nectin-based cell-cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities

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-3alpha (biggest), -3beta (middle), and -3gamma (smallest). Like nectin-1 and -2, nectin-3alpha 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-3alpha formed a cis-homo-dimer and showed Ca(2+)-independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3alpha 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-hetero-interaction with nectin-2. The affinity of trans-hetero-interaction of nectin-3alpha with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3alpha. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3alpha was colocalized with nectin-2 at cadherin-based 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.     

Vav2 as a Rac-GDP/GTP exchange factor responsible for the nectin-induced, c-Src- and Cdc42-mediated activation of Rac

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that form homo- and hetero-trans-dimers (trans-interactions). Nectins first form cell-cell contact and then recruit cadherins to the nectin-based cell-cell contact sites to form adherens junctions cooperatively with cadherins. In addition, the trans-interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which enhances the formation of adherens junctions by forming filopodia and lamellipodia, respectively. The trans-interactions of nectins first recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then phosphorylates and activates FRG, a Cdc42-GDP/GTP exchange factor (GEF) for Cdc42. The activation of both c-Src and Cdc42 by FRG is necessary for the activation of Rac, but the Rac-GEF responsible for this activation of Rac remains unknown. We showed here that the nectin-induced activation of Rac was inhibited by a dominant negative mutant of Vav2, a Rac-GEF. Nectins recruited and tyrosine-phosphorylated Vav2 through c-Src at the nectin-based cell-cell contact sites, whereas Cdc42 was not necessary for the nectin-induced recruitment of Vav2 or the nectin-induced, c-Src-mediated tyrosine phosphorylation of Vav2. Cdc42 activated through c-Src then enhanced the GEF activity of tyrosine-phosphorylated Vav2 on Rac1. These results indicate that Vav2 is a GEF responsible for the nectin-induced, c-Src-, and Cdc42-mediated activation of Rac.     

α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.     

Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.     

Requirement of interaction of nectin-1alpha/HveC with afadin for efficient cell-cell spread of herpes simplex virus type 1

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 alpha-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-1alpha 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-1alpha capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1alpha incapable of interacting with afadin, we found that the interaction of nectin-1alpha with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1alpha 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-1alpha at cadherin-based AJs.     

Nectins and nectin-like molecules: roles in cell adhesion, polarization, movement, and proliferation

Nectins and nectin-like molecules (Necls) are immunoglobulin-like cell adhesion molecules that constitute families containing four and five members, respectively. All members, except for Necl-5, trans-interact homophilically. Furthermore, all members, including Necl-5, trans-interact heterophilically with their respective specific partners among the members. Necl-5 regulates cell movement and proliferation cooperatively with integrin alphavbeta3 and growth factor receptors. Nectins function as cell-cell adhesion molecules at a variety of cell-cell junctions, including adherens junctions, and regulate the initial step of cell-cell junction formation. Nectins and integrin alphavbeta3 are further involved in the cross-talk between cell-matrix and cell-cell junctions. Thus, both nectin and Necl family members play important roles in fundamental cellular functions, including cell adhesion, polarization, movement, and proliferation.     

Recruitment of nectin-3 to cell-cell junctions through trans-heterophilic interaction with CD155, a vitronectin and poliovirus receptor that localizes to alpha(v)beta3 integrin-containing membrane microdomains

Nectins present a novel class of Ig superfamily adhesion molecules that, cooperatively with cadherins, establish and maintain cell-cell adherens junctions. CD155, the cognate receptor for poliovirus, undergoes cell-matrix contacts by binding to the extracellular matrix protein vitronectin. The significant homology of nectins with CD155 prompted us to investigate the possibility of their interaction. We determined that nectin-3 binds CD155 and its putative mouse homologue Tage4 in cell-based ligand binding assays. Coculture of nectin-3- and CD155-expressing HeLa cells led to CD155-dependent recruitment of nectin-3 to cell-cell contacts. In a heterologous coculture system with CD155 expressing mouse neuroblastoma cells, HeLa cell-expressed nectin-3 was recruited to contact sites with CD155 bearing neurites. CD155 and nectin-3 colocalized to epithelial cell-cell junctions in renal proximal tubules and in the amniotic membrane. Efficient interaction depended on CD155 dimerization, which appears to be aided by cell type-specific cofactors. We furthermore found CD155 to codistribute with alpha(v) integrin microdomains on the surface of transfected mouse fibroblasts and at amniotic epithelial cell junctions. Our findings demonstrate the possible trans-interaction between the bona fide cell-cell adherens type adhesion system (cadherin/nectin) and the cell-matrix adhesion system (integrin/CD155) by virtue of their nectin-3 and CD155 components, respectively.     

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and alpha-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound alpha-actinin, an actin filament-bundling protein, which bound to alpha-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and alpha-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through alpha-actinin.     

Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.     

Roles and modes of action of nectins in cell–cell adhesion

Nectins are Ca²⁺-independent immunoglobulin (Ig)-like cell–cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell–cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell–cell junctions and cell–cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell–cell adhesion and recruit E-cadherin to the nectin-based cell–cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

Involvement of the annexin II-S100A10 complex in the formation of E-cadherin-based adherens junctions in Madin-Darby canine kidney cells

E-cadherin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca(2+), E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca(2+), E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca(2+), E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca(2+) in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca(2+), E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-cadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the nectin-based cell-cell contact sites in the Ca(2+) switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.