title: Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.
abstract: ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with alpha catenin, but not to that with beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin was approximately 0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of approximately 10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.
authors.affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Itoh
authors.forename: A
authors.initials: A
authors.lastname: Nagafuchi
authors.forename: S
authors.initials: S
authors.lastname: Moroi
authors.forename: S
authors.initials: S
authors.lastname: Tsukita
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D007739:L Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051178:alpha Catenin; D051176:beta Catenin
chemical_list: D000199:Actins; C495267:CTNNB1 protein, mouse; D015820:Cadherins; C495272:Ctnna1 protein, mouse; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051178:alpha Catenin; D051176:beta Catenin