Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein - PubMed (original) (raw)

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein

K Takahashi et al. J Cell Biol. 1999.

Abstract

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

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Figures

Figure 1

Figure 1

Structures of the full length and various fragments of l-afadin and nectin. PR, proline-rich region; ABD, F-actin–binding domain; TM, transmembrane region.

Figure 2

Figure 2

Direct binding of l-afadin to nectin in vitro. (A) Affinity chromatography. GST-l-afadin-PDZ (aa 1007–1125) or GST alone (20 μg of protein each) was applied to protein A–Sepharose beads on which Myc-nectin-1-CP (aa 379–518) was immobilized through the anti-Myc mAb. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. Arrow, GST-l-afadin-PDZ; arrowhead, Myc-nectin-1-CP. (B) 35S-Labeled l-afadin blot overlay. GST-fusion proteins of the cytoplasmic regions of nectin (0.3 μg of protein each) were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue or by 35S-labeled l-afadin blot overlay. (B1) Protein staining. (B2) 35S-Labeled l-afadin blot overlay. GST-Nectin-1-CPC, aa 449–518; GST-Nectin-1-CPC-ΔC, aa 449–514; GST-Nectin-2α-CP, aa 387–467; GST-Nectin-2α-CP-ΔC, aa 387–463; GST-Nectin-2δ-CP, aa 403–530; GST-Nectin-2δ-CP-ΔC, aa 403–526.

Figure 3

Figure 3

Coimmunoprecipitation of l-afadin and nectin. (A) Immunoprecipitation from the bile canaliculi-rich fraction of mouse liver. An extract from the bile canaliculi-rich fraction was subjected to immunoprecipitation with the anti–nectin-2 mAb or control IgG. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti–l-afadin mAb or the anti–nectin-2α pAb. (B) Immunoprecipitation from mouse mammary tumor MTD-1A cells. A cell extract from MTD-1A cells was subjected to immunoprecipitation with the anti–nectin-2 mAb or control IgG. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti–l-afadin mAb, the anti–nectin-2α pAb, or the anti– nectin-2δ pAb. (C) Immunoprecipitation from COS7 cells expressing FLAG-nectin-1 and/or Myc-l-afadin. FLAG-Nectin-1 alone, Myc-l-afadin alone, or both the proteins were transiently expressed in COS7 cells. Each cell extract was subjected to immunoprecipitation with the anti-FLAG or anti-Myc mAb. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti-FLAG or anti-Myc mAb. IP, immunoprecipitation.

Figure 4

Figure 4

Localization of nectin, l-afadin, and E-cadherin in epithelial cells. (A) Localization sites of nectin-2, l-afadin, and E-cadherin in mouse small intestine. The frozen sections of mouse small intestine were triply stained with the rat anti–nectin-2 mAb, the rabbit anti–l-afadin pAb, and the mouse anti–E-cadherin mAb. They were visualized with rhodamine-conjugated anti– rat IgG, FITC-conjugated anti– rabbit IgG, and Cy5-conjugated anti–mouse IgG Abs. (A1) Nectin-2; (A2) l-afadin; (A3) E-cadherin. Asterisks mark the inner space of small intestine. Bar, 50 μm. (B) Localization sites of nectin-1, l-afadin, and E-cadherin in MDCK cells. MDCK cells stably expressing FLAG-nectin-1 were triply stained with the mouse anti-FLAG mAb, the rabbit anti–l-afadin pAb, and the rat anti–E-cadherin mAb. They were visualized with FITC-conjugated anti–mouse IgG, rhodamine-conjugated anti–rabbit IgG, and Cy5-conjugated anti–rat IgG Abs. (B1 and B4) FLAG-nectin-1; (B2 and B5) l-afadin; (B3 and B6) E-cadherin; (B1–B3) junction-level view; (B4–B6) cross-sectional view. The cross-sectional view was generated by confocal microscopy. Arrow, apical level; arrowhead, basal level. Bars, 10 μm.

Figure 5

Figure 5

Ultrastructural localization sites of nectin-2 in mouse small intestine absorptive epithelial cells. Intestine absorptive epithelial cells were labeled with the anti–nectin-2 mAb using the silver-enhancement technique. Open arrow, ZO; closed arrow, ZA; asterisk, desmosome. Bars, 0.2 μm.

Figure 6

Figure 6

Localization of nectin and l-afadin in nonepithelial cells. (A) Localization sites of nectin-2 and l-afadin in mouse heart. The frozen sections of mouse heart were doubly stained with the rat anti–nectin-2 mAb and the rabbit anti–l-afadin pAb. They were visualized with rhodamine-conjugated anti–rat IgG and FITC-conjugated anti–rabbit IgG Abs. (A1) Nectin-2; (A2) l-afadin. Arrows, intercalated disc. Bar, 10 μm. (B) Localization sites of nectin-2 and l-afadin in cultured EL cells. EL cells were doubly stained with the rat anti–nectin-2 mAb and the mouse anti–l-afadin mAb. They were visualized with FITC-conjugated anti–rat IgG and rhodamine-conjugated anti–mouse IgG Abs. There was nuclear staining with this anti–l-afadin mAb, but its significance is not clear. (B1) Nectin-2; (B2) l-afadin. Bar, 10 μm.

Figure 7

Figure 7

Recruitment of nectin to cadherin-based AJs through interaction with l-afadin. (A) Immunoprecipitation from EL cells expressing FLAG-nectin-1 or FLAG-nectin-1-ΔC. FLAG-Nectin-1 or FLAG-nectin-1-ΔC was transiently expressed in EL cells. Each cell extract was subjected to immunoprecipitation with the anti-FLAG mAb. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti-FLAG or anti–l-afadin mAb. (B) Immunofluorescence microscopy of FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells. FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells were doubly stained with the mouse anti-FLAG mAb and the rat anti–E-cadherin mAb or the rabbit anti–l-afadin pAb. They were visualized with FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rat or anti–rabbit IgG Abs. There was nuclear staining with this anti–l-afadin pAb, but its significance is not clear. (B1–B6) FLAG-nectin-1-EL cells; (B7–B12) FLAG-nectin-1-ΔC-EL cells; (B1 and B4) FLAG-nectin-1; (B7 and B10) FLAG-nectin-1-ΔC; (B2 and B8) E-cadherin; (B5 and B11) l-afadin; (B3, B6, B9, and B12) merge. Arrows, cell–cell AJs. Bars, 10 μm.

Figure 7

Figure 7

Recruitment of nectin to cadherin-based AJs through interaction with l-afadin. (A) Immunoprecipitation from EL cells expressing FLAG-nectin-1 or FLAG-nectin-1-ΔC. FLAG-Nectin-1 or FLAG-nectin-1-ΔC was transiently expressed in EL cells. Each cell extract was subjected to immunoprecipitation with the anti-FLAG mAb. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti-FLAG or anti–l-afadin mAb. (B) Immunofluorescence microscopy of FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells. FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells were doubly stained with the mouse anti-FLAG mAb and the rat anti–E-cadherin mAb or the rabbit anti–l-afadin pAb. They were visualized with FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rat or anti–rabbit IgG Abs. There was nuclear staining with this anti–l-afadin pAb, but its significance is not clear. (B1–B6) FLAG-nectin-1-EL cells; (B7–B12) FLAG-nectin-1-ΔC-EL cells; (B1 and B4) FLAG-nectin-1; (B7 and B10) FLAG-nectin-1-ΔC; (B2 and B8) E-cadherin; (B5 and B11) l-afadin; (B3, B6, B9, and B12) merge. Arrows, cell–cell AJs. Bars, 10 μm.

Figure 8

Figure 8

Cell aggregation activity of nectin. (A) Ca2+-independent aggregation activity of nectin-1 and -2α. L cells stably expressing nectin-1 or nectin-2α were treated with trypsin in the presence of EDTA and then dispersed by pipetting to obtain a single-cell suspension. Each single-cell suspension was rotated in HBSS in the presence of 1 mM CaCl2 or 1 mM EDTA for 15, 30, and 60 min. The extent of aggregation of cells was represented by the ratio of the total particle number at time t of incubation (Nt) to the initial particle number (No). (A1) Nectin-1-L cells. (Filled circles and open circles) Wild-type L cells; (filled triangles and open triangles) nectin-1-L cells; (filled circles and filled triangles) in the presence of 1 mM CaCl2; and (open circles and open triangles) in the presence of 1 mM EDTA. (A2) Nectin-2α-L cells. (Filled circles and open circles) Wild-type L cells; (filled triangles and open triangles) nectin-2α-L cells; (filled circles and filled triangles) in the presence of 1 mM CaCl2; and (open circles and open triangles) in the presence of 1 mM EDTA. The Nt/No values are the means ± SD of three independent experiments. (B) Cell aggregation of nectin-1-L cells. Single cells were rotated in HBSS in the presence of 1 mM EDTA for 60 min. (B1) Wild-type L cells. (B2) Nectin-1-L cells. Bars, 100 μm. These results are representative of three independent experiments.

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References

    1. Albelda SM, Buck CA. Integrin and other cell adhesion molecules. FASEB (Fed Am Soc Exp Biol) J. 1990;4:2868–2880. - PubMed
    1. Aoki J, Koike S, Ise I, Sato-Yoshida Y, Nomoto A. Amino acid residues on human poliovirus receptor involved in interaction with poliovirus. J Biol Chem. 1994;269:8431–8438. - PubMed
    1. Aoki J, Koike S, Asou H, Ise I, Suwa H, Tanaka T, Miyasaka M, Nomoto A. Mouse homolog of poliovirus receptor-related gene 2 product, mPRR2, mediates homophilic cell aggregation. Exp Cell Res. 1997;235:374–384. - PubMed
    1. Barth AIM, Näthke IS, Nelson WJ. Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol. 1997;9:683–690. - PubMed
    1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. - PubMed

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