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Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Vaidyanath A, Hashizume T, Nagaoka T, Takeyasu N, Satoh H, Chen L, Wang J, Kasai T, Kudoh T, Satoh A, Fu L, Seno M - J. Cell. Mol. Med. (2011)

Bottom Line: Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand.Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells.Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nano-Biotechnology, Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Kita-ku, Okayama, Japan.

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Assessment of the mechanism of internalization of EC-Fc/BNC in SK-BR-3 cells. (A) SK-BR-3 cells and SK-OV-3 cells were stained with antibodies against ErbB2 (green) and Cav-1 (red). (B) SK-BR-3 cells were treated with EC-Fc/BNC in the presence or absence of 100 nM of CPZ and stained with anti-human IgG antibody labelled with FITC. Transferrin-RITC was used as a control for the internalization. (C) SK-BR-3 cells were treated with EC-Fc/BNC labelled with RITC in the presence or absence of mβCD. The cells were stained with anti–EEA-1 antibody followed by secondary antibody against mouse IgG labelled with AlexaFlour-488. The cells were then stripped with and without acid treatment to remove the surface bound fraction and to visualize the internalized fraction. Bars, 10 μm.
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fig05: Assessment of the mechanism of internalization of EC-Fc/BNC in SK-BR-3 cells. (A) SK-BR-3 cells and SK-OV-3 cells were stained with antibodies against ErbB2 (green) and Cav-1 (red). (B) SK-BR-3 cells were treated with EC-Fc/BNC in the presence or absence of 100 nM of CPZ and stained with anti-human IgG antibody labelled with FITC. Transferrin-RITC was used as a control for the internalization. (C) SK-BR-3 cells were treated with EC-Fc/BNC labelled with RITC in the presence or absence of mβCD. The cells were stained with anti–EEA-1 antibody followed by secondary antibody against mouse IgG labelled with AlexaFlour-488. The cells were then stripped with and without acid treatment to remove the surface bound fraction and to visualize the internalized fraction. Bars, 10 μm.

Mentions: Cells adopt divergent pathways for the endocytosis of the cargos and receptors. The key pathways were categorized into clathrin-dependent and clathrin-independent mechanisms [33]. The clathrin-independent pathway is further classified into caveolar and GPI-anchored early endocytic compartments (GEEC) pathways [33, 34]. ErbB2 is thought to be internalizing in SK-OV-3 through clathrin-mediated internalization [24]. In this study we found that the mechanism of internalization of ErbB2 present in SK-OV-3 cells is deficient in SK-BR-3 cells. We tried to identify the difference between the two cell lines using DNA microarray (Fig. S2). As a result the expression of claudin 16, caveolin-1 and caveolin-2 were found to be extensively down-regulated in SK-BR-3 cells when compared to SK-OV-3 cells. This absence of caveolin-1 in SK-BR-3 cells was further confirmed by immunostaining using anti-Cav1 antibody (Fig. 5A). SK-BR-3 did not show the presence of caveolin-1 suggesting that the impaired caveolar mechanism prevailing in the cell, which was consistent with the finding by the previous reports [13, 35]. Because caveolin-1 was detected in SK-OV-3 cells the internalization pathway deficient in SK-BR-3 cells might be attributed to caveolae.


Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Vaidyanath A, Hashizume T, Nagaoka T, Takeyasu N, Satoh H, Chen L, Wang J, Kasai T, Kudoh T, Satoh A, Fu L, Seno M - J. Cell. Mol. Med. (2011)

Assessment of the mechanism of internalization of EC-Fc/BNC in SK-BR-3 cells. (A) SK-BR-3 cells and SK-OV-3 cells were stained with antibodies against ErbB2 (green) and Cav-1 (red). (B) SK-BR-3 cells were treated with EC-Fc/BNC in the presence or absence of 100 nM of CPZ and stained with anti-human IgG antibody labelled with FITC. Transferrin-RITC was used as a control for the internalization. (C) SK-BR-3 cells were treated with EC-Fc/BNC labelled with RITC in the presence or absence of mβCD. The cells were stained with anti–EEA-1 antibody followed by secondary antibody against mouse IgG labelled with AlexaFlour-488. The cells were then stripped with and without acid treatment to remove the surface bound fraction and to visualize the internalized fraction. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822962&req=5

fig05: Assessment of the mechanism of internalization of EC-Fc/BNC in SK-BR-3 cells. (A) SK-BR-3 cells and SK-OV-3 cells were stained with antibodies against ErbB2 (green) and Cav-1 (red). (B) SK-BR-3 cells were treated with EC-Fc/BNC in the presence or absence of 100 nM of CPZ and stained with anti-human IgG antibody labelled with FITC. Transferrin-RITC was used as a control for the internalization. (C) SK-BR-3 cells were treated with EC-Fc/BNC labelled with RITC in the presence or absence of mβCD. The cells were stained with anti–EEA-1 antibody followed by secondary antibody against mouse IgG labelled with AlexaFlour-488. The cells were then stripped with and without acid treatment to remove the surface bound fraction and to visualize the internalized fraction. Bars, 10 μm.
Mentions: Cells adopt divergent pathways for the endocytosis of the cargos and receptors. The key pathways were categorized into clathrin-dependent and clathrin-independent mechanisms [33]. The clathrin-independent pathway is further classified into caveolar and GPI-anchored early endocytic compartments (GEEC) pathways [33, 34]. ErbB2 is thought to be internalizing in SK-OV-3 through clathrin-mediated internalization [24]. In this study we found that the mechanism of internalization of ErbB2 present in SK-OV-3 cells is deficient in SK-BR-3 cells. We tried to identify the difference between the two cell lines using DNA microarray (Fig. S2). As a result the expression of claudin 16, caveolin-1 and caveolin-2 were found to be extensively down-regulated in SK-BR-3 cells when compared to SK-OV-3 cells. This absence of caveolin-1 in SK-BR-3 cells was further confirmed by immunostaining using anti-Cav1 antibody (Fig. 5A). SK-BR-3 did not show the presence of caveolin-1 suggesting that the impaired caveolar mechanism prevailing in the cell, which was consistent with the finding by the previous reports [13, 35]. Because caveolin-1 was detected in SK-OV-3 cells the internalization pathway deficient in SK-BR-3 cells might be attributed to caveolae.

Bottom Line: Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand.Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells.Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nano-Biotechnology, Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Kita-ku, Okayama, Japan.

Show MeSH
Related in: MedlinePlus