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Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions.

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.

Animals↗

Herpes simplex virus entry receptor nectin-1 is widely expressed in the murine eye.

PURPOSE: Nectin-1 belongs to the immunoglobulin superfamily, mediates cell-cell adhesion in cadherin-based adherens junctions, and acts as a receptor for herpes simplex virus (HSV). The goals of this study were (1) to determine whether nectin-1 is expressed in ocular tissue that is an important target of HSV infections and (2) to determine whether HSV type 1 (HSV-1) infection affects nectin-1 expression in the eye. METHODS: Expression of nectin-1 and HSV-1 protein was determined by immunohistochemical analysis of ocular tissues of untreated BALB/c mice and mice that were euthanized either 7 days or 7 months after corneal inoculation of HSV-1 or sterile tissue-culture medium (mock). RESULTS: In ocular tissues derived from untreated and mock-infected mice, widespread nectin-1 expression was detected among cells of the corneal epithelium and endothelium, conjunctiva, lens epithelium, ciliary body, iris, choroid, and retina. However, fibroblasts in the corneal stroma and the sclera did not express detectable levels of nectin-1. Ocular tissues from mice euthanized 7 days after corneal inoculation of HSV-1 frequently demonstrated corneal ulceration and inflammation and HSV-1 protein expression in the corneal epithelium, stroma, endothelium, conjunctiva, iris, and ciliary body but rarely in the retina. Ocular tissues from mice euthanized 7 months after HSV-1 inoculation demonstrated corneal epithelial and stromal inflammation, but HSV-1 protein expression was not detected. HSV-1 infection did not lead to a loss of nectin-1 expression in any of the tissues examined. In contrast to uninfected corneas, the inflamed and vascularized stroma of infected corneas contained mononuclear inflammatory cells, vascular cells, and fibroblasts that stained positive for nectin-1. CONCLUSIONS: Findings report that nectin-1 is widely expressed in murine ocular tissues. Only fibroblasts in the corneal stroma and sclera of uninfected tissues were devoid of nectin-1 expression. HSV-1-infected inflamed corneas contained some stromal fibroblasts with detectable nectin-1 expression, which potentially could be targeted by the virus. Widespread nectin-1 expression in the eye suggests that this receptor may play a role in the pathogenesis of ocular HSV infections.

Animals↗

Expression of nectin-2 in mouse granulosa cells.

OBJECTIVE: The development and maturation of the ovarian follicles are characterized by structural changes that require components involved in cell-cell adhesion systems. Recently a novel group of cell adhesion molecules named nectins has been identified. The present study examined expression and cell-specific localization of nectins during mouse follicular development. STUDY DESIGN: Expression of nectins in mouse ovary was investigated by immnuoblot analysis and immunohistochemistry. More precise localization was determined by electron microscopy. RESULTS: Immunoblot analysis revealed expression of nectin-2 and nectin-3 but not nectin-1 in ovarian granulosa cells. Immunohistochemistry demonstrated expression of nectin-2 at cell-cell adhesion sites of granulosa cell layer in the primary and preantral follicles. Especially, intense immunoreactivity of nectin-2 accumulated around the zona pellucida. In antral follicles, the intensity of nectin-2 expression on granulosa cells was decreased. By electron microscopy nectin-2 was detected not only on thin extensions of granulosa cells penetrating the zona pellucida, but also on the attachment sites between thin extensions of granulosa cells and oocyte surface. CONCLUSION: The restricted expression of nectin-2 in the granulosa cells of primary and preantral follicles might reflect some of the molecular changes in cell-cell adhesion during early follicular development.

Animals↗

Nectin-dependent localization of synaptic scaffolding molecule (S-SCAM) at the puncta adherentia junctions formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the mouse hippocampus.

BACKGROUND: Two types of intercellular junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAs), are observed at the synapses between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the hippocampus. SJs are associated with active zones and postsynaptic densities (PSDs) where neurotransmission occurs, whereas PAs are not associated with either of them. We have found that the nectin-afadin unit as well as the N-cadherin-catenin unit localizes at the PAs and that both the units cooperatively organize the PAs. Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Synaptic scaffolding molecule (S-SCAM) is a neural scaffolding protein which interacts with many proteins including neuroligin, NMDA receptors, neural plakophilin-related armadillo-repeat protein/delta-catenin, a GDP/GTP exchange protein for Rap1 small G protein (PDZ-Rap-GEP), and beta-catenin. S-SCAM has been suggested to be a component of PSDs, but its precise localization at the synapses remains unknown. RESULTS: S-SCAM was not concentrated at the PSDs but highly concentrated and co-localized with nectins at both the sides of the PAs formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the adult mouse hippocampus. S-SCAM co-localized with nectin-1 at the primitive synapses where the SJs and the PAs were not morphologically differentiated, and they co-localized during the maturation of the SJs and the PAs. Nectin-1 had a potency to recruit S-SCAM to the nectin-1-based cell-cell adhesion sites formed in cadherin-deficient L cells as a model system. This recruitment was dependent on the C-terminal PDZ domain-binding motif of nectin-1 which is necessary for the binding of afadin, suggesting that nectins recruit S-SCAM through afadin. Consistently, S-SCAM was co-immunoprecipitated with afadin by the anti-S-SCAM antibody from the mouse brain, but S-SCAM did not directly bind afadin. CONCLUSION: These results indicate that S-SCAM localizes at the PAs in the CA3 area of the hippocampus in a nectin-dependent manner and suggest that S-SCAM serves as a scaffolding molecule at the PAs after maturation of the synapses and at the SJs during the maturation.

Adaptor Proteins, Signal Transducing↗

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.

Animals↗

Structural features of nectin-2 (HveB) required for herpes simplex virus entry.

One step in the process of herpes simplex virus (HSV) entry into cells is the binding of viral glycoprotein D (gD) to a cellular receptor. Human nectin-2 (also known as HveB and Prr2), a member of the immunoglobulin (Ig) superfamily, serves as a gD receptor for the entry of HSV-2, variant forms of HSV-1 that have amino acid substitutions at position 25 or 27 of gD (for example, HSV-1/Rid), and porcine pseudorabies virus (PRV). The gD binding region of nectin-2 is believed to be localized to the N-terminal variable-like (V) Ig domain. In order to identify specific amino acid sequences in nectin-2 that are important for HSV entry activity, chimeric molecules were constructed by exchange of sequences between human nectin-2 and its mouse homolog, mouse nectin-2, which mediates entry of PRV but not HSV-1 or HSV-2. The nectin-2 chimeric molecules were expressed in Chinese hamster ovary cells, which normally lack a gD receptor, and tested for cell surface expression and viral entry activity. As expected, chimeric molecules containing the V domain of human nectin-2 exhibited HSV entry activity. Replacement of either of two small regions in the V domain of mouse nectin-2 with amino acids from the equivalent positions in human nectin-2 (amino acids 75 to 81 or 89) transferred HSV-1/Rid entry activity to mouse nectin-2. The resulting chimeras also exhibited enhanced HSV-2 entry activity and gained the ability to mediate wild-type HSV-1 entry. Replacement of amino acid 89 of human nectin-2 with the corresponding mouse amino acid (M89F) eliminated HSV entry activity. These results identify two different amino acid sequences, predicted to lie adjacent to the C' and C" beta-strands of the V domain, that are critical for HSV entry activity. This region is homologous to the human immunodeficiency virus binding region of CD4 and to the poliovirus binding region of CD155.

Amino Acid Sequence↗

Nectin-2-mediated entry of a syncytial strain of herpes simplex virus via pH-independent fusion with the plasma membrane of Chinese hamster ovary cells.

BACKGROUND: Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway. RESULTS: Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH-independent entry at the plasma membrane. In addition, CHO-nectin-2 cells supported pH-dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid (t1/2 of 5-10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO. CONCLUSION: HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.

Animals↗

Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus.

Nectin and afadin constitute a novel intercellular adhesion system that organizes adherens junctions in cooperation with the cadherin-catenin system in epithelial cells. Nectin is a Ca(2+)-independent immunoglobulin-like adhesion molecule and afadin is an actin filament (F-actin)-binding protein that connects nectin to the actin cytoskeleton. At the puncta adhaerentia junctions (PAs) between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, the nectin-afadin system also colocalizes with the cadherin-catenin system and has a role in the formation of synapses. ZO-1 is another F-actin-binding protein that localizes at tight junctions (TJs) and connects claudin to the actin cytoskeleton in epithelial cells. The nectin-afadin system is able to recruit ZO-1 to the nectin-based cell-cell adhesion sites in nonepithelial cells that have no TJs. In the present study, we investigated the localization of ZO-1 in the mouse hippocampus. Immunofluorescence and immunoelectron microscopy revealed that ZO-1 also localized at the PAs between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, as described for afadin. ZO-1 colocalized with afadin during the development of synaptic junctions and PAs. Microbeads coated with the extracellular fragment of nectin, which interacts with cellular nectin, recruited both afadin and ZO-1 to the bead-cell contact sites in cultured rat hippocampal neurons. These results indicate that ZO-1 colocalizes with nectin and afadin at the PAs and that the nectin-afadin system is involved in the localization of ZO-1.

Animals↗

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Amino Acid Sequence↗

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.

Animals↗

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.

Actins↗

Mutations in herpes simplex virus glycoprotein D that prevent cell entry via nectins and alter cell tropism.

Glycoprotein D (gD) determines which cells can be infected by herpes simplex virus (HSV) by binding to one of the several cell surface receptors that can mediate HSV entry or cell fusion. These receptors include the herpesvirus entry mediator (HVEM), nectin-1, nectin-2, and sites in heparan sulfate generated by specific 3-O-sulfotransferases. The objective of the present study was to identify residues in gD that are critical for physical and functional interactions with nectin-1 and nectin-2. We found that double or triple amino acid substitutions at positions 215, 222, and 223 in gD caused marked reduction in gD binding to nectin-1 and a corresponding inability to function in cell fusion or entry of HSV via nectin-1 or nectin-2. These substitutions either enhanced or did not significantly inhibit functional interactions with HVEM and modified heparan sulfate. These and other results demonstrate that different domains of gD, with some overlap, are critical for functional interactions with each class of entry receptor. Viral entry assays, using gD mutants described here and previously, revealed that nectins are the principal entry receptors for selected human cell lines of neuronal and epithelial origin, whereas HVEM or nectins could be used to mediate entry into a T lymphocyte line. Because T cells and fibroblasts can be infected via HVEM, HSV strains carrying gD mutations that prevent entry via nectins may establish transient infections in humans, but perhaps not latent infections of neurons, and are therefore candidates for development of safe live virus vaccines and vaccine vectors.

Animals↗

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.

Actins↗

A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions.

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non-trans-interacting nectins), inhibited the E-cadherin-induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non-trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin-induced activation of Rac and formation of cell-cell AJs.

Adherens Junctions↗

Recruitment of E-cadherin associated with alpha- and beta-catenins and p120ctn to the nectin-based cell-cell adhesion sites by the action of 12-O-tetradecanoylphorbol-13-acetate in MDCK cells.

The formation of tight junctions (TJs) is dependent on the formation of adherens junctions (AJs) in MDCK cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin, occludin and junctional adhesion molecule (JAM) are major CAMs at TJs. When MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 mm Ca(2+), nectin first forms cell-cell adhesion and recruits E-cadherin to the nectin-based cell-cell adhesion sites to form AJs. Thereafter, nectin recruits first JAM-A and then claudin-1 and occludin to the apical side of AJs to form TJs. In contrast, when MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 microm Ca(2+) in the presence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a TJ-like structure is formed without the formation of the E-cadherin-based AJs. We showed here that GFP-E-cadherin, which did not trans-interact due to 2 microm Ca(2+) but associated with alpha- and beta-catenins and p120(ctn), was recruited to the nectin-based cell-cell adhesion sites by the action of TPA. The nectin inhibitors, which inhibited the trans-interaction of nectin, inhibited the recruitment of GFP-E-cadherin and their associating catenins by the action of TPA. Microbeads coated with the extracellular fragment of nectin recruited not only cellular nectin but also GFP-E-cadherin and their associating catenins by the action of TPA. These results indicate that when the TJ-like structure is formed by the action of TPA, non-trans-interacting E-cadherin and its associating catenins are recruited to the nectin-based cell-cell adhesion sites and that the trans-interaction of E-cadherin is not essential for the formation of TJs.

Animals↗

Nectin-like molecule-1/TSLL1/SynCAM3: a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes.

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules and comprise a family of four members. At the mossy fiber terminals of hippocampus, nectin-1 and nectin-3 localize at the presynaptic and postsynaptic sides of synaptic junctions, respectively, and their trans-interactions play a role in formation of synapses in cooperation with N-cadherin. Nectins are associated with the actin cytoskeleton through afadin, a nectin- and actin-filament-binding protein. Five nectin-like molecules (Necls) which have domain structures similar to those of nectins have been identified and here we characterize Necl-1/TSLL1/SynCAM3, from now on referred to as Necl-1. Tissue distribution analysis showed that Necl-1 was specifically expressed in the neural tissue. Immunofluorescence and immunoelectron microscopy revealed that Necl-1 localized at the contact sites among axons, their terminals, and glia cell processes that cooperatively formed synapses, axon bundles and myelinated axons. Necl-1 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 from now on referred to as Necl-2, nectin-1 and nectin-3, but not with Necl-5 or nectin-2. The C-terminal cytoplasmic region of Necl-1 did not bind afadin but bound membrane-associated guanylate kinase subfamily members that contain the L27 domain, including Dlg3, Pals2 and CASK. These results indicate that Necl-1 is a neural-tissue-specific Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which potentially has membrane-associated guanylate kinase subfamily member-binding activity and localizes at the non-junctional cell-cell contact sites.

Actins↗

Nectin-2 expression in testicular cells is controlled via the functional cooperation between transcription factors of the Sp1, CREB, and AP-1 families.

Nectin-2, a major protein component of the adherens junctions (AJs), is found between Sertoli cells and germ cells in the seminiferous epithelium. Recent studies have shown that the expression of nectin-2 gene in testis is crucial to maintain normal spermatogenesis since male knockout mice lacking nectin-2 gene are sterile and possess morphologically abnormal spermatozoa. However, the molecular mechanisms governing its basal transcription remain poorly understood. By the use of Sertoli and germ cell-lines (TM4 and GC-2spd(ts) cells, respectively) in transient transfection studies, we showed that the minimal mouse nectin-2 promoter was located between nucleotides -316 and -211 (relative to the translation start site). Two putative Sp1 motifs and one each of the CRE, AP1, and AP2 motifs were identified within this region. Mutational studies showed that these two Sp1 motifs cooperated synergistically with the CRE motif, but not the AP1 and AP2 motifs, to regulate nectin-2 gene transcription in both TM4 and GC-2spd(ts) cells. By EMSAs, we found that an AP-1 consensus sequence was able to inhibit DNA-protein complex formation with the CRE motif, suggesting an interaction between the AP-1 transcription factor (c-Jun) and CREB within the CRE motif. Overexpressions of CREB and c-Jun, but not c-Fos, also significantly increased the promoter activity, which suggests that CREB and c-Jun are the crucial transcription factors involved in regulating nectin-2 gene transcription. Chromatin immunoprecipitation assay has shown that, in vivo, CREB, c-Jun, and Sp1 family proteins are bound to the mouse nectin-2 promoter. Analysis of the staged tubules has confirmed that the cyclic expressions of CREB and nectin-2 coincide with the event of adherens junction restructuring between Sertoli cells and germ cells. The cross-talk between CREB, c-Jun, and Sp1 family protein is believed to be a major transcription machinery to drive nectin-2 expression in Sertoli cells.

Animals↗

Nectin-1 expression by squamous cell carcinoma is a predictor of herpes oncolytic sensitivity.

Oncolytic viruses based on herpes simplex virus type 1 (HSV-1) are able to infect and lyse a variety of malignant cell lines. However, there is variability in the degree of tumor susceptibility, and the cancer cell determinants of HSV sensitivity are poorly defined. Nectin-1 is a cell surface adhesion molecule that functions as a cellular receptor to HSV envelope glycoprotein D (gD). We assessed tumor nectin-1 expression as a predictor of oncolytic HSV sensitivity. A panel of human squamous carcinoma cell lines was evaluated for viral entry, replication, and cytotoxicity to an attenuated, replication-competent, oncolytic HSV (NV1023). Potential tumor determinants of HSV sensitivity were assessed, including nectin-1, herpes viral entry mediator, total gD receptor expression, S-phase fraction, and doubling time. Significant correlations between nectin-1 expression measured by quantitative fluorescence-activated cell sorting and viral sensitivity measures were identified using Pearson's coefficients. Cancer cell nectin-1 receptor blockade and nectin-1 transfection led to inhibition and enhancement of NV1023 viral entry, respectively. Cell lines with varying nectin-1 expression showed corresponding sensitivity to NV1023 therapy in vivo. Immunohistochemistry for nectin-1 was inversely related to E-cadherin staining, suggesting increased herpes sensitivity of E-cadherin-deficient tumors. These results suggest that nectin-1 may be used as a marker to predict the sensitivity of a tumor to herpes oncolytic therapy.

Animals↗