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Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Fujito T, Ikeda W, Kakunaga S, Minami Y, Kajita M, Sakamoto Y, Monden M, Takai Y - J. Cell Biol. (2005)

Bottom Line: This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis.Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells.These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan.

ABSTRACT
Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

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Clathrin-dependent endocytosis of Necl-5. (A) Inhibition of down-regulation of Necl-5 by a DN mutant of epsin, Eps15, or dynamin, but not by a DN mutant of caveolin. Immunofluorescence images of the cells transfected with various DN mutants. The cells were transfected with myc-epsin DN mutant (a), EGFP-Eps15 DN mutant (b), HA-dynamin DN mutant (c), or EGFP-caveolin DN mutant (d); cultured at the higher density; and double or triple stained with various combinations of the anti–Necl-5 mAb-i, the anti-myc mAb, the anti-HA mAb, and the anti–nectin-3 pAb. Asterisks show the transfected cells. Bars, 10 μm. (B) Assembly of clathrin heavy chain at the cytoplasmic region of Necl-5, which interacts with Nef-3. NIH3T3 cells were cultured at the lower density and then cultured in the presence or absence of Nef-3– or IgG-coated beads for 1 h. The cells were double stained with the anti–Necl-5 mAb-i and the anticlathrin heavy chain mAb. (a) In the presence of the Nef-3–coated beads; (b) in the presence of the IgG-coated beads; (c) in the absence of the beads. Asterisks, the positions of the beads; insets, magnified images of the boxed areas. Bars, 10 μm. The results shown are representative of three independent experiments.
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fig4: Clathrin-dependent endocytosis of Necl-5. (A) Inhibition of down-regulation of Necl-5 by a DN mutant of epsin, Eps15, or dynamin, but not by a DN mutant of caveolin. Immunofluorescence images of the cells transfected with various DN mutants. The cells were transfected with myc-epsin DN mutant (a), EGFP-Eps15 DN mutant (b), HA-dynamin DN mutant (c), or EGFP-caveolin DN mutant (d); cultured at the higher density; and double or triple stained with various combinations of the anti–Necl-5 mAb-i, the anti-myc mAb, the anti-HA mAb, and the anti–nectin-3 pAb. Asterisks show the transfected cells. Bars, 10 μm. (B) Assembly of clathrin heavy chain at the cytoplasmic region of Necl-5, which interacts with Nef-3. NIH3T3 cells were cultured at the lower density and then cultured in the presence or absence of Nef-3– or IgG-coated beads for 1 h. The cells were double stained with the anti–Necl-5 mAb-i and the anticlathrin heavy chain mAb. (a) In the presence of the Nef-3–coated beads; (b) in the presence of the IgG-coated beads; (c) in the absence of the beads. Asterisks, the positions of the beads; insets, magnified images of the boxed areas. Bars, 10 μm. The results shown are representative of three independent experiments.

Mentions: There are at least two types of endocytosis: clathrin-dependent and -independent ones (Conner and Schmid, 2003). Epsin and Eps15 are regulatory components of the formation of clathrin-coated vesicles (Conner and Schmid, 2003), and the ENTH (epsin NH2-terminal homology domain) and the DIII domain of Eps15 act as dominant-negative (DN) mutants for the clathrin-dependent endocytosis (Benmerah et al., 1998; Nakashima et al., 1999). Dynamin is an important regulator for the formation of both clathrin-dependent and -independent endocytic vesicles, and its DN mutant, dynamin1 K44A, inhibits various types of endocytosis (Conner and Schmid, 2003). Caveolin regulates the formation of caveolae and its endocytosis, and an NH2-terminal–truncated mutant of caveolin, DGV-caveolin, serves as a DN mutant and inhibits the formation of caveolae by perturbing intracellular cholesterol trafficking (Conner and Schmid, 2003). To test whether or not the endocytosis of Necl-5 depends on clathrin, various DN mutants were expressed in NIH3T3 cells, and the cells were plated and cultured at the higher density. The nectin-3–induced decrease of the cell surface signal for Necl-5 was inhibited by the expression of the myc-tagged epsin DN mutant (Fig. 4 A, a1–a4, asterisks). The signal for nectin-3 did not change. The essentially similar results were obtained by the expression of the EGFP-tagged Eps15 DN mutant or HA-tagged dynamin DN mutant (Fig. 4 A, b1–c4, asterisks). The nectin-3–induced decrease of the cell surface signal for Necl-5 was not affected by the expression of the EGFP-tagged caveolin DN mutant (Fig. 4 A, d1–d4, asterisks). These results indicate that Necl-5 is down-regulated by the clathrin-dependent endocytosis.


Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3.

Fujito T, Ikeda W, Kakunaga S, Minami Y, Kajita M, Sakamoto Y, Monden M, Takai Y - J. Cell Biol. (2005)

Clathrin-dependent endocytosis of Necl-5. (A) Inhibition of down-regulation of Necl-5 by a DN mutant of epsin, Eps15, or dynamin, but not by a DN mutant of caveolin. Immunofluorescence images of the cells transfected with various DN mutants. The cells were transfected with myc-epsin DN mutant (a), EGFP-Eps15 DN mutant (b), HA-dynamin DN mutant (c), or EGFP-caveolin DN mutant (d); cultured at the higher density; and double or triple stained with various combinations of the anti–Necl-5 mAb-i, the anti-myc mAb, the anti-HA mAb, and the anti–nectin-3 pAb. Asterisks show the transfected cells. Bars, 10 μm. (B) Assembly of clathrin heavy chain at the cytoplasmic region of Necl-5, which interacts with Nef-3. NIH3T3 cells were cultured at the lower density and then cultured in the presence or absence of Nef-3– or IgG-coated beads for 1 h. The cells were double stained with the anti–Necl-5 mAb-i and the anticlathrin heavy chain mAb. (a) In the presence of the Nef-3–coated beads; (b) in the presence of the IgG-coated beads; (c) in the absence of the beads. Asterisks, the positions of the beads; insets, magnified images of the boxed areas. Bars, 10 μm. The results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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fig4: Clathrin-dependent endocytosis of Necl-5. (A) Inhibition of down-regulation of Necl-5 by a DN mutant of epsin, Eps15, or dynamin, but not by a DN mutant of caveolin. Immunofluorescence images of the cells transfected with various DN mutants. The cells were transfected with myc-epsin DN mutant (a), EGFP-Eps15 DN mutant (b), HA-dynamin DN mutant (c), or EGFP-caveolin DN mutant (d); cultured at the higher density; and double or triple stained with various combinations of the anti–Necl-5 mAb-i, the anti-myc mAb, the anti-HA mAb, and the anti–nectin-3 pAb. Asterisks show the transfected cells. Bars, 10 μm. (B) Assembly of clathrin heavy chain at the cytoplasmic region of Necl-5, which interacts with Nef-3. NIH3T3 cells were cultured at the lower density and then cultured in the presence or absence of Nef-3– or IgG-coated beads for 1 h. The cells were double stained with the anti–Necl-5 mAb-i and the anticlathrin heavy chain mAb. (a) In the presence of the Nef-3–coated beads; (b) in the presence of the IgG-coated beads; (c) in the absence of the beads. Asterisks, the positions of the beads; insets, magnified images of the boxed areas. Bars, 10 μm. The results shown are representative of three independent experiments.
Mentions: There are at least two types of endocytosis: clathrin-dependent and -independent ones (Conner and Schmid, 2003). Epsin and Eps15 are regulatory components of the formation of clathrin-coated vesicles (Conner and Schmid, 2003), and the ENTH (epsin NH2-terminal homology domain) and the DIII domain of Eps15 act as dominant-negative (DN) mutants for the clathrin-dependent endocytosis (Benmerah et al., 1998; Nakashima et al., 1999). Dynamin is an important regulator for the formation of both clathrin-dependent and -independent endocytic vesicles, and its DN mutant, dynamin1 K44A, inhibits various types of endocytosis (Conner and Schmid, 2003). Caveolin regulates the formation of caveolae and its endocytosis, and an NH2-terminal–truncated mutant of caveolin, DGV-caveolin, serves as a DN mutant and inhibits the formation of caveolae by perturbing intracellular cholesterol trafficking (Conner and Schmid, 2003). To test whether or not the endocytosis of Necl-5 depends on clathrin, various DN mutants were expressed in NIH3T3 cells, and the cells were plated and cultured at the higher density. The nectin-3–induced decrease of the cell surface signal for Necl-5 was inhibited by the expression of the myc-tagged epsin DN mutant (Fig. 4 A, a1–a4, asterisks). The signal for nectin-3 did not change. The essentially similar results were obtained by the expression of the EGFP-tagged Eps15 DN mutant or HA-tagged dynamin DN mutant (Fig. 4 A, b1–c4, asterisks). The nectin-3–induced decrease of the cell surface signal for Necl-5 was not affected by the expression of the EGFP-tagged caveolin DN mutant (Fig. 4 A, d1–d4, asterisks). These results indicate that Necl-5 is down-regulated by the clathrin-dependent endocytosis.

Bottom Line: This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis.Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells.These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan.

ABSTRACT
Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor-induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell-cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell-cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell-cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.

Show MeSH
Related in: MedlinePlus