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

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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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