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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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Association of N-ZO-1, N-ZO-2, and full-length ZO-3 with the cytoplasmic domain of claudin-1 in vitro. The GST fusion protein with the cytoplasmic domain of claudin-1 (GST-Cln-1) or GST protein (GST), which was bound to glutathione–Sepharose beads, was incubated with the lysate of Sf9 cells expressing N-ZO-1, C-ZO-1, 6xHis-N-ZO-2, or 6xHis-C-ZO-2, or with the lysate of E. coli expressing 6xHis-tagged full-length ZO-3. In each lysate, the amount of recombinant protein was adjusted to be the same. After washing, the proteins associated with GST fusion protein or GST were eluted from the beads with a buffer containing glutathione. The eluates from N-ZO-1–, C-ZO-1–, 6xHis-N-ZO-2–, 6xHis-C-ZO-2–, or 6xHis-ZO-3–incubated beads were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb T8754, anti–ZO-1 mAb T7713, anti-His tag mAb, anti-His tag mAb, or anti-His tag mAb (immunoblotting). The amount of GST–claudin-1 (GST-claudin-1) and GST (GST) in each eluate was determined by Coomassie brilliant blue staining (CBB). As indicated by arrowheads, N-ZO-1, N-ZO-2, full-length ZO-3, but not C-ZO-1 or C-ZO-2, showed binding affinity to the cytoplasmic domain of claudin-1. Bars indicate molecular masses of 200, 116, 97, 66, 45, and 31 kD, respectively, from the top.
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Figure 3: Association of N-ZO-1, N-ZO-2, and full-length ZO-3 with the cytoplasmic domain of claudin-1 in vitro. The GST fusion protein with the cytoplasmic domain of claudin-1 (GST-Cln-1) or GST protein (GST), which was bound to glutathione–Sepharose beads, was incubated with the lysate of Sf9 cells expressing N-ZO-1, C-ZO-1, 6xHis-N-ZO-2, or 6xHis-C-ZO-2, or with the lysate of E. coli expressing 6xHis-tagged full-length ZO-3. In each lysate, the amount of recombinant protein was adjusted to be the same. After washing, the proteins associated with GST fusion protein or GST were eluted from the beads with a buffer containing glutathione. The eluates from N-ZO-1–, C-ZO-1–, 6xHis-N-ZO-2–, 6xHis-C-ZO-2–, or 6xHis-ZO-3–incubated beads were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb T8754, anti–ZO-1 mAb T7713, anti-His tag mAb, anti-His tag mAb, or anti-His tag mAb (immunoblotting). The amount of GST–claudin-1 (GST-claudin-1) and GST (GST) in each eluate was determined by Coomassie brilliant blue staining (CBB). As indicated by arrowheads, N-ZO-1, N-ZO-2, full-length ZO-3, but not C-ZO-1 or C-ZO-2, showed binding affinity to the cytoplasmic domain of claudin-1. Bars indicate molecular masses of 200, 116, 97, 66, 45, and 31 kD, respectively, from the top.

Mentions: As shown in Fig. 2, various constructs for ZO-1, ZO-2, and ZO-3 were used in this study for in vitro biding assays and in vivo colocalization analyses. First, we produced recombinant N-ZO-1, C-ZO-1, N-ZO-2 (6xHis tag), and C-ZO-2 (6xHis tag) in Sf9 cells by baculovirus infection (Itoh et al. 1997, Itoh et al. 1999), and full-length ZO-3 in E. coli, and their cell lysates containing the same amount of recombinant proteins were mixed with beads conjugated with the GST-fusion protein with the cytoplasmic domain of claudin-1. Bound proteins were then eluted from beads and each eluate was subjected to SDS-PAGE followed by immunoblotting with corresponding antibodies. As shown in Fig. 3N-ZO-1, N-ZO-2, and full-length ZO-3, but not C-ZO-1 or C-ZO-2, appeared to specifically bind to the GST-fusion protein with the cytoplasmic domain of claudin-1.


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)

Association of N-ZO-1, N-ZO-2, and full-length ZO-3 with the cytoplasmic domain of claudin-1 in vitro. The GST fusion protein with the cytoplasmic domain of claudin-1 (GST-Cln-1) or GST protein (GST), which was bound to glutathione–Sepharose beads, was incubated with the lysate of Sf9 cells expressing N-ZO-1, C-ZO-1, 6xHis-N-ZO-2, or 6xHis-C-ZO-2, or with the lysate of E. coli expressing 6xHis-tagged full-length ZO-3. In each lysate, the amount of recombinant protein was adjusted to be the same. After washing, the proteins associated with GST fusion protein or GST were eluted from the beads with a buffer containing glutathione. The eluates from N-ZO-1–, C-ZO-1–, 6xHis-N-ZO-2–, 6xHis-C-ZO-2–, or 6xHis-ZO-3–incubated beads were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb T8754, anti–ZO-1 mAb T7713, anti-His tag mAb, anti-His tag mAb, or anti-His tag mAb (immunoblotting). The amount of GST–claudin-1 (GST-claudin-1) and GST (GST) in each eluate was determined by Coomassie brilliant blue staining (CBB). As indicated by arrowheads, N-ZO-1, N-ZO-2, full-length ZO-3, but not C-ZO-1 or C-ZO-2, showed binding affinity to the cytoplasmic domain of claudin-1. Bars indicate molecular masses of 200, 116, 97, 66, 45, and 31 kD, respectively, from the top.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168087&req=5

Figure 3: Association of N-ZO-1, N-ZO-2, and full-length ZO-3 with the cytoplasmic domain of claudin-1 in vitro. The GST fusion protein with the cytoplasmic domain of claudin-1 (GST-Cln-1) or GST protein (GST), which was bound to glutathione–Sepharose beads, was incubated with the lysate of Sf9 cells expressing N-ZO-1, C-ZO-1, 6xHis-N-ZO-2, or 6xHis-C-ZO-2, or with the lysate of E. coli expressing 6xHis-tagged full-length ZO-3. In each lysate, the amount of recombinant protein was adjusted to be the same. After washing, the proteins associated with GST fusion protein or GST were eluted from the beads with a buffer containing glutathione. The eluates from N-ZO-1–, C-ZO-1–, 6xHis-N-ZO-2–, 6xHis-C-ZO-2–, or 6xHis-ZO-3–incubated beads were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb T8754, anti–ZO-1 mAb T7713, anti-His tag mAb, anti-His tag mAb, or anti-His tag mAb (immunoblotting). The amount of GST–claudin-1 (GST-claudin-1) and GST (GST) in each eluate was determined by Coomassie brilliant blue staining (CBB). As indicated by arrowheads, N-ZO-1, N-ZO-2, full-length ZO-3, but not C-ZO-1 or C-ZO-2, showed binding affinity to the cytoplasmic domain of claudin-1. Bars indicate molecular masses of 200, 116, 97, 66, 45, and 31 kD, respectively, from the top.
Mentions: As shown in Fig. 2, various constructs for ZO-1, ZO-2, and ZO-3 were used in this study for in vitro biding assays and in vivo colocalization analyses. First, we produced recombinant N-ZO-1, C-ZO-1, N-ZO-2 (6xHis tag), and C-ZO-2 (6xHis tag) in Sf9 cells by baculovirus infection (Itoh et al. 1997, Itoh et al. 1999), and full-length ZO-3 in E. coli, and their cell lysates containing the same amount of recombinant proteins were mixed with beads conjugated with the GST-fusion protein with the cytoplasmic domain of claudin-1. Bound proteins were then eluted from beads and each eluate was subjected to SDS-PAGE followed by immunoblotting with corresponding antibodies. As shown in Fig. 3N-ZO-1, N-ZO-2, and full-length ZO-3, but not C-ZO-1 or C-ZO-2, appeared to specifically bind to the GST-fusion protein with the cytoplasmic domain of claudin-1.

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