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Involvement of nectin in the localization of IQGAP1 at the cell–cell adhesion sites through the actin cytoskeleton in Madin–Darby canine kidney cells

Oncogene volume 22, pages 2097–2109 (2003)Cite this article

Abstract

IQGAP1, a putative downstream target of the Rho family small G proteins, Cdc42 and Rac, localizes at adherens junctions (AJs) in epithelial cells. It has been suggested that IQGAP1 localizes at AJs through its binding to β-catenin, and negatively regulates the E-cadherin-mediated cell–cell adhesion. Nectin is a Ca2+-independent, immunoglobulin-like cell–cell adhesion molecule that localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs cooperatively with E-cadherin. Here we investigated a role of nectin in the localization of IQGAP1 at AJs. Ca2+ chelation from the medium causes disruption of the E-cadherin-mediated cell–cell adhesion, but not the nectin-based cell–cell adhesion, in Madin–Darby canine kidney (MDCK) cells. IQGAP1 remained at the residual nectin-based cell–cell adhesion sites where the E-cadherin immunofluorescence signal disappeared. Restoration of Ca2+ in the medium causes re-accumulation of E-cadherin to the residual nectin-based cell–cell adhesion sites to re-form AJs. Nectin inhibitors inhibit this re-accumulation of E-cadherin to re-form AJs by impairing the nectin-based cell–cell adhesion. The nectin inhibitors also reduced the localization of IQGAP1 at the cell–cell adhesion sites. When MDCK cells were incubated with microbeads coated with the extracellular fragment of nectin that interacts with cellular nectin, IQGAP1 also accumulated at the bead–MDCK cell contact sites. The accumulation of IQGAP1 at the cell–cell adhesion sites was inhibited by actin filament-disrupting agents, latrunculin A and cytochalasin D. These results indicate that nectin is involved in the localization of IQGAP1 at AJs through the actin cytoskeleton.

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Abbreviations

aa:

amino acid(s)

Ab:

antibody

AJs:

adherens junctions

BSA:

bovine serum albumin

DSP:

dithiobis succinimidylpropionate

gD:

fragment of glycoprotein D fused to IgG Fc

mAb:

monoclonal antibody

MDCK:

Madin–Darby canine kidney

Nef-3:

extracellular fragment of nectin-3 fused to IgG Fc

pAb:

polyclonal antibody

PAGE:

polyacrylamide gel electrophoresis

PBS:

phosphate-buffered saline

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Acknowledgements

We thank Dr A Nagafuchi (Kumamoto University, Kumamoto, Japan) for valuable discussions and critical readings of the manuscript, Dr M Takeichi (Center for Developmental Biology, RIKEN, Kobe, Japan) for providing us with the anti-E-cadherin mAb, and Dr W Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. This investigation was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, Culture, and Technology, Japan (2001, 2002).

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

    Tatsuo Katata, Kenji Irie, Atsunori Fukuhara, Tomomi Kawakatsu, Akio Yamada, Kazuya Shimizu & Yoshimi Takai

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  1. Tatsuo Katata
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Correspondence to Yoshimi Takai.

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Katata, T., Irie, K., Fukuhara, A. et al. Involvement of nectin in the localization of IQGAP1 at the cell–cell adhesion sites through the actin cytoskeleton in Madin–Darby canine kidney cells. Oncogene 22, 2097–2109 (2003). https://doi.org/10.1038/sj.onc.1206255

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