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Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S - J. Cell Biol. (1999)

Bottom Line: Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

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Recruitment of exogenous ZO-2 to claudin-based networks in L transfectants expressing claudin-1. Full-length ZO-2 (full-length; a and b), deletion mutant of ZO-2 lacking both PDZ1 and -2 domains (ΔPDZ1,2; c and d), deletion mutant of ZO-2 lacking PDZ1 domain (ΔPDZ1; e and f), and deletion mutant of ZO-2 lacking PDZ2 domain (ΔPDZ2; g and h) were transfected into L transfectants expressing claudin-1 (C1L cells), and stable transfectants were obtained. Furthermore, full-length ZO-2 (full-length; i and j) was transfected into L transfectants expressing claudin-1 mutant lacking its COOH-terminal YV sequence (C1ΔYVL cell), and stable transfectants were obtained. These introduced proteins were tagged with c-myc epitope. These stable transfectants were double stained with anti–claudin-1 mAb (a, c, e, and g) or pAb (i) and anti–c-myc mAb (b, d, f, h, and j). In C1L cells where claudin-1 was concentrated at cell–cell borders in an elaborate network pattern, full-length ZO-2 (b), ΔPDZ1-ZO-2 (f), and ΔPDZ2-ZO-2 (h), but not ΔPDZ1,2-ZO-2 (d), were recruited to the claudin-1–based networks. Insets represent the network patterns of concentrated claudin-1 (a and g), full-length ZO-2 (b), and ΔPDZ2-ZO-2 (h). No concentration of full-length ZO-2 was observed in C1ΔYVL cells (j). Bar: (a–j) 10 μm; (insets) 15 μm.
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Figure 8: Recruitment of exogenous ZO-2 to claudin-based networks in L transfectants expressing claudin-1. Full-length ZO-2 (full-length; a and b), deletion mutant of ZO-2 lacking both PDZ1 and -2 domains (ΔPDZ1,2; c and d), deletion mutant of ZO-2 lacking PDZ1 domain (ΔPDZ1; e and f), and deletion mutant of ZO-2 lacking PDZ2 domain (ΔPDZ2; g and h) were transfected into L transfectants expressing claudin-1 (C1L cells), and stable transfectants were obtained. Furthermore, full-length ZO-2 (full-length; i and j) was transfected into L transfectants expressing claudin-1 mutant lacking its COOH-terminal YV sequence (C1ΔYVL cell), and stable transfectants were obtained. These introduced proteins were tagged with c-myc epitope. These stable transfectants were double stained with anti–claudin-1 mAb (a, c, e, and g) or pAb (i) and anti–c-myc mAb (b, d, f, h, and j). In C1L cells where claudin-1 was concentrated at cell–cell borders in an elaborate network pattern, full-length ZO-2 (b), ΔPDZ1-ZO-2 (f), and ΔPDZ2-ZO-2 (h), but not ΔPDZ1,2-ZO-2 (d), were recruited to the claudin-1–based networks. Insets represent the network patterns of concentrated claudin-1 (a and g), full-length ZO-2 (b), and ΔPDZ2-ZO-2 (h). No concentration of full-length ZO-2 was observed in C1ΔYVL cells (j). Bar: (a–j) 10 μm; (insets) 15 μm.

Mentions: Since ZO-2 was not expressed endogenously in L cells (see Fig. 6), the full-length cDNA encoding c-myc–tagged ZO-2 was introduced into C1L cells. As shown in Fig. 8, a and b, this introduced ZO-2 was recruited to the claudin-based networks. However, since ZO-2 can directly bind to ZO-1 through its PDZ2 domain (Itoh et al. 1999), it was not clear whether this recruitment of full-length ZO-2 was based on the direct interaction of ZO-2 with claudin-1 or endogenous ZO-1 that was recruited to the claudin-based networks as shown in Fig. 7. Therefore, we next transfected cDNAs encoding c-myc–tagged ZO-2 mutant lacking both PDZ1 and -2 (ΔPDZ1,2-ZO-2), PDZ1 alone (ΔPDZ1-ZO-2) or PDZ2 alone (ΔPDZ2-ZO-2) into L transfectants (see Fig. 2), confirmed their expression by immunoblotting, and their subcellular localization was followed by anti–c-myc mAb staining. ΔPDZ1,2-ZO-2 was not recruited to the claudin-based networks (Fig. 8c and Fig. d), whereas ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 were recruited (Fig. 8, e–h). These findings can be interpreted as indicating that ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 are recruited by the direct association with endogenous ZO-1 (through PDZ2) and claudin-1 (through PDZ1), respectively. Since ΔPDZ1,2-ZO-2 cannot bind to either endogenous ZO-1 or claudin-1, it would not be recruited. In good agreement with this, in C1ΔYVL cells, exogenous full-length ZO-2 was not concentrated at cell–cell borders, probably because in these cells endogenous ZO-1 as well as exogenous ZO-2 cannot bind to mutant claudin-1 (Fig. 8i and Fig. j). The same results were obtained when C2L cells were used (data not shown).


Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S - J. Cell Biol. (1999)

Recruitment of exogenous ZO-2 to claudin-based networks in L transfectants expressing claudin-1. Full-length ZO-2 (full-length; a and b), deletion mutant of ZO-2 lacking both PDZ1 and -2 domains (ΔPDZ1,2; c and d), deletion mutant of ZO-2 lacking PDZ1 domain (ΔPDZ1; e and f), and deletion mutant of ZO-2 lacking PDZ2 domain (ΔPDZ2; g and h) were transfected into L transfectants expressing claudin-1 (C1L cells), and stable transfectants were obtained. Furthermore, full-length ZO-2 (full-length; i and j) was transfected into L transfectants expressing claudin-1 mutant lacking its COOH-terminal YV sequence (C1ΔYVL cell), and stable transfectants were obtained. These introduced proteins were tagged with c-myc epitope. These stable transfectants were double stained with anti–claudin-1 mAb (a, c, e, and g) or pAb (i) and anti–c-myc mAb (b, d, f, h, and j). In C1L cells where claudin-1 was concentrated at cell–cell borders in an elaborate network pattern, full-length ZO-2 (b), ΔPDZ1-ZO-2 (f), and ΔPDZ2-ZO-2 (h), but not ΔPDZ1,2-ZO-2 (d), were recruited to the claudin-1–based networks. Insets represent the network patterns of concentrated claudin-1 (a and g), full-length ZO-2 (b), and ΔPDZ2-ZO-2 (h). No concentration of full-length ZO-2 was observed in C1ΔYVL cells (j). Bar: (a–j) 10 μm; (insets) 15 μm.
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Figure 8: Recruitment of exogenous ZO-2 to claudin-based networks in L transfectants expressing claudin-1. Full-length ZO-2 (full-length; a and b), deletion mutant of ZO-2 lacking both PDZ1 and -2 domains (ΔPDZ1,2; c and d), deletion mutant of ZO-2 lacking PDZ1 domain (ΔPDZ1; e and f), and deletion mutant of ZO-2 lacking PDZ2 domain (ΔPDZ2; g and h) were transfected into L transfectants expressing claudin-1 (C1L cells), and stable transfectants were obtained. Furthermore, full-length ZO-2 (full-length; i and j) was transfected into L transfectants expressing claudin-1 mutant lacking its COOH-terminal YV sequence (C1ΔYVL cell), and stable transfectants were obtained. These introduced proteins were tagged with c-myc epitope. These stable transfectants were double stained with anti–claudin-1 mAb (a, c, e, and g) or pAb (i) and anti–c-myc mAb (b, d, f, h, and j). In C1L cells where claudin-1 was concentrated at cell–cell borders in an elaborate network pattern, full-length ZO-2 (b), ΔPDZ1-ZO-2 (f), and ΔPDZ2-ZO-2 (h), but not ΔPDZ1,2-ZO-2 (d), were recruited to the claudin-1–based networks. Insets represent the network patterns of concentrated claudin-1 (a and g), full-length ZO-2 (b), and ΔPDZ2-ZO-2 (h). No concentration of full-length ZO-2 was observed in C1ΔYVL cells (j). Bar: (a–j) 10 μm; (insets) 15 μm.
Mentions: Since ZO-2 was not expressed endogenously in L cells (see Fig. 6), the full-length cDNA encoding c-myc–tagged ZO-2 was introduced into C1L cells. As shown in Fig. 8, a and b, this introduced ZO-2 was recruited to the claudin-based networks. However, since ZO-2 can directly bind to ZO-1 through its PDZ2 domain (Itoh et al. 1999), it was not clear whether this recruitment of full-length ZO-2 was based on the direct interaction of ZO-2 with claudin-1 or endogenous ZO-1 that was recruited to the claudin-based networks as shown in Fig. 7. Therefore, we next transfected cDNAs encoding c-myc–tagged ZO-2 mutant lacking both PDZ1 and -2 (ΔPDZ1,2-ZO-2), PDZ1 alone (ΔPDZ1-ZO-2) or PDZ2 alone (ΔPDZ2-ZO-2) into L transfectants (see Fig. 2), confirmed their expression by immunoblotting, and their subcellular localization was followed by anti–c-myc mAb staining. ΔPDZ1,2-ZO-2 was not recruited to the claudin-based networks (Fig. 8c and Fig. d), whereas ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 were recruited (Fig. 8, e–h). These findings can be interpreted as indicating that ΔPDZ1-ZO-2 and ΔPDZ2-ZO-2 are recruited by the direct association with endogenous ZO-1 (through PDZ2) and claudin-1 (through PDZ1), respectively. Since ΔPDZ1,2-ZO-2 cannot bind to either endogenous ZO-1 or claudin-1, it would not be recruited. In good agreement with this, in C1ΔYVL cells, exogenous full-length ZO-2 was not concentrated at cell–cell borders, probably because in these cells endogenous ZO-1 as well as exogenous ZO-2 cannot bind to mutant claudin-1 (Fig. 8i and Fig. j). The same results were obtained when C2L cells were used (data not shown).

Bottom Line: Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

Show MeSH
Related in: MedlinePlus