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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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Behavior of GFP-fusion proteins with PDZ1, -2, and -3 domains of ZO-1/ZO-2/ZO-3 in cultured epithelial cells (MDCK cells). GFP (GFP) or GFP-fusion proteins with PDZ1 domain of ZO-1 (P1-ZO-1-GFP), PDZ2 domain of ZO-1 (P2-ZO-1-GFP), PDZ3 domain of ZO-1 (P3-ZO-1-GFP), PDZ1 domain of ZO-2 (P1-ZO-2-GFP) or PDZ1 domain of ZO-3 (P1-ZO-3-GFP) were exogenously and transiently expressed in MDCK cells. These cells were fixed and stained with anti–claudin-1 pAb (a, c, e, g, i, and k) in red. Expressed GFP or GFP-fusion proteins were visualized by green fluorescence (b, d, f, h, j, and l). PDZ1 domains of ZO-1 (b, P1-ZO-1-GFP), ZO-2 (h, P1-ZO-2-GFP), ZO-3 (j, P1-ZO-3-GFP), and PDZ2 domain of ZO-1 (d, P2-ZO-1-GFP) were recruited to claudin-1–positive TJs. PDZ3 domain of ZO-1 (f, P3-ZO-1-GFP) also appeared to be concentrated at TJs, although very faintly as compared with b, d, h, and j. No concentration of GFP at cell–cell borders was observed (l). Bar, 10 μm.
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Figure 10: Behavior of GFP-fusion proteins with PDZ1, -2, and -3 domains of ZO-1/ZO-2/ZO-3 in cultured epithelial cells (MDCK cells). GFP (GFP) or GFP-fusion proteins with PDZ1 domain of ZO-1 (P1-ZO-1-GFP), PDZ2 domain of ZO-1 (P2-ZO-1-GFP), PDZ3 domain of ZO-1 (P3-ZO-1-GFP), PDZ1 domain of ZO-2 (P1-ZO-2-GFP) or PDZ1 domain of ZO-3 (P1-ZO-3-GFP) were exogenously and transiently expressed in MDCK cells. These cells were fixed and stained with anti–claudin-1 pAb (a, c, e, g, i, and k) in red. Expressed GFP or GFP-fusion proteins were visualized by green fluorescence (b, d, f, h, j, and l). PDZ1 domains of ZO-1 (b, P1-ZO-1-GFP), ZO-2 (h, P1-ZO-2-GFP), ZO-3 (j, P1-ZO-3-GFP), and PDZ2 domain of ZO-1 (d, P2-ZO-1-GFP) were recruited to claudin-1–positive TJs. PDZ3 domain of ZO-1 (f, P3-ZO-1-GFP) also appeared to be concentrated at TJs, although very faintly as compared with b, d, h, and j. No concentration of GFP at cell–cell borders was observed (l). Bar, 10 μm.

Mentions: A question naturally arose as to whether PDZ1 domains of ZO-1/ZO-2/ZO-3 interact with the cytoplasmic domain of claudins in epithelial cells. To evaluate this point, we constructed expression vectors for green fluorescent protein (GFP)-fusion proteins with PDZ1, PDZ2, or PDZ3 domains of ZO-1 and introduced them into cultured MDCK cells (Fig. 10, a–f). For an unknown reason, all these fusion proteins were concentrated in the nucleus. In addition to the nuclear staining, PDZ1-GFP and PDZ2-GFP were clearly recruited to and concentrated at claudin-1–positive TJs (Fig. 10, a–d). Similarly to L transfectants, these findings can be interpreted as indicating that PDZ1-GFP and PDZ2-GFP are recruited by the direct association with endogenous claudins and ZO-2/ZO-3, respectively. In epithelial cells, PDZ3-GFP also appeared to be concentrated at TJs, although very faintly as compared with PDZ1-GFP and PDZ2-GFP (Fig. 10e and Fig. f), probably due to unidentified binding partners for PDZ3 domain of ZO-1 localized at TJs in epithelial cells. GFP-fusion proteins with PDZ1 (Fig. 10, g–j) or PDZ2 (data not shown) domains of ZO-2/ZO-3 also showed significant concentration at claudin-1–positive TJs in MDCK cells.


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)

Behavior of GFP-fusion proteins with PDZ1, -2, and -3 domains of ZO-1/ZO-2/ZO-3 in cultured epithelial cells (MDCK cells). GFP (GFP) or GFP-fusion proteins with PDZ1 domain of ZO-1 (P1-ZO-1-GFP), PDZ2 domain of ZO-1 (P2-ZO-1-GFP), PDZ3 domain of ZO-1 (P3-ZO-1-GFP), PDZ1 domain of ZO-2 (P1-ZO-2-GFP) or PDZ1 domain of ZO-3 (P1-ZO-3-GFP) were exogenously and transiently expressed in MDCK cells. These cells were fixed and stained with anti–claudin-1 pAb (a, c, e, g, i, and k) in red. Expressed GFP or GFP-fusion proteins were visualized by green fluorescence (b, d, f, h, j, and l). PDZ1 domains of ZO-1 (b, P1-ZO-1-GFP), ZO-2 (h, P1-ZO-2-GFP), ZO-3 (j, P1-ZO-3-GFP), and PDZ2 domain of ZO-1 (d, P2-ZO-1-GFP) were recruited to claudin-1–positive TJs. PDZ3 domain of ZO-1 (f, P3-ZO-1-GFP) also appeared to be concentrated at TJs, although very faintly as compared with b, d, h, and j. No concentration of GFP at cell–cell borders was observed (l). Bar, 10 μm.
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Figure 10: Behavior of GFP-fusion proteins with PDZ1, -2, and -3 domains of ZO-1/ZO-2/ZO-3 in cultured epithelial cells (MDCK cells). GFP (GFP) or GFP-fusion proteins with PDZ1 domain of ZO-1 (P1-ZO-1-GFP), PDZ2 domain of ZO-1 (P2-ZO-1-GFP), PDZ3 domain of ZO-1 (P3-ZO-1-GFP), PDZ1 domain of ZO-2 (P1-ZO-2-GFP) or PDZ1 domain of ZO-3 (P1-ZO-3-GFP) were exogenously and transiently expressed in MDCK cells. These cells were fixed and stained with anti–claudin-1 pAb (a, c, e, g, i, and k) in red. Expressed GFP or GFP-fusion proteins were visualized by green fluorescence (b, d, f, h, j, and l). PDZ1 domains of ZO-1 (b, P1-ZO-1-GFP), ZO-2 (h, P1-ZO-2-GFP), ZO-3 (j, P1-ZO-3-GFP), and PDZ2 domain of ZO-1 (d, P2-ZO-1-GFP) were recruited to claudin-1–positive TJs. PDZ3 domain of ZO-1 (f, P3-ZO-1-GFP) also appeared to be concentrated at TJs, although very faintly as compared with b, d, h, and j. No concentration of GFP at cell–cell borders was observed (l). Bar, 10 μm.
Mentions: A question naturally arose as to whether PDZ1 domains of ZO-1/ZO-2/ZO-3 interact with the cytoplasmic domain of claudins in epithelial cells. To evaluate this point, we constructed expression vectors for green fluorescent protein (GFP)-fusion proteins with PDZ1, PDZ2, or PDZ3 domains of ZO-1 and introduced them into cultured MDCK cells (Fig. 10, a–f). For an unknown reason, all these fusion proteins were concentrated in the nucleus. In addition to the nuclear staining, PDZ1-GFP and PDZ2-GFP were clearly recruited to and concentrated at claudin-1–positive TJs (Fig. 10, a–d). Similarly to L transfectants, these findings can be interpreted as indicating that PDZ1-GFP and PDZ2-GFP are recruited by the direct association with endogenous claudins and ZO-2/ZO-3, respectively. In epithelial cells, PDZ3-GFP also appeared to be concentrated at TJs, although very faintly as compared with PDZ1-GFP and PDZ2-GFP (Fig. 10e and Fig. f), probably due to unidentified binding partners for PDZ3 domain of ZO-1 localized at TJs in epithelial cells. GFP-fusion proteins with PDZ1 (Fig. 10, g–j) or PDZ2 (data not shown) domains of ZO-2/ZO-3 also showed significant concentration at claudin-1–positive TJs in MDCK cells.

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