Honda Tomoyuki, Shimizu Kazuya, Kawakatsu Tomomi, Yasumi Masato, Shingai Tatsushi, Fukuhara Atsunori, Ozaki-Kuroda Kumi, Irie Kenji, Nakanishi Hiroyuki, Takai Yoshimi
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
Genes Cells. 2003 Jan;8(1):51-63. doi: 10.1046/j.1365-2443.2003.00616.x.
Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at the E-cadherin-based cell-cell adherens junctions (AJs), and comprises a family consisting of four members, nectin-1, -2, -3, and -4. Nectin and E-cadherin are associated with afadin and alpha-catenin, actin filament (F-actin)-binding proteins connecting respective adhesion molecules to the actin cytoskeleton, but the role of nectin in the formation of the E-cadherin-based cell-cell AJs has not yet been fully understood. To obtain evidence for this role of nectin, we attempted to develop an antagonist and/or agonist of nectin.
We made a recombinant extracellular fragment of nectin-3 (Nef-3). Nef-3 trans-interacted with cellular nectin-1 and thereby diminished the formation of the nectin-1-based cell-cell adhesion. This resulted in a reduction of the formation of the E-cadherin-based cell-cell adhesion in L fibroblasts stably expressing both exogenous nectin-1alpha and E-cadherin (nectin-1-EL cells) and MDCK cells stably expressing exogenous nectin-1alpha (nectin-1-MDCK cells). This antagonistic effect of Nef-3 was also observed in L cells stably expressing exogenous E-cadherin alone (EL cells) and wild-type MDCK cells. Conversely, Nef-3 coated on microbeads first recruited the nectin-afadin complex and then the E-cadherin-catenin complex to the bead-cell contact sites in nectin-1-EL and nectin-1-MDCK cells.
These results suggest that nectin is necessary and sufficient for the recruitment of E-cadherin to the nectin-based cell-cell adhesion sites and involved in the formation of E-cadherin-based cell-cell AJs.
Nectin是一种基于E-钙黏蛋白的细胞间黏附连接（AJs）处不依赖Ca2+的免疫球蛋白样细胞间黏附分子，由四个成员组成一个家族，即Nectin-1、-2、-3和-4。Nectin和E-钙黏蛋白与afadin和α-连环蛋白相关，后者是将各自的黏附分子连接到肌动蛋白细胞骨架的肌动蛋白丝（F-肌动蛋白）结合蛋白，但Nectin在基于E-钙黏蛋白的细胞间AJs形成中的作用尚未完全明确。为了获得Nectin这一作用的证据，我们试图开发Nectin的拮抗剂和/或激动剂。
我们制备了重组的Nectin-3细胞外片段（Nef-3）。Nef-3与细胞表面的Nectin-1发生反式相互作用，从而减少了基于Nectin-1的细胞间黏附的形成。这导致在稳定表达外源性Nectin-1α和E-钙黏蛋白的L成纤维细胞（Nectin-1-EL细胞）以及稳定表达外源性Nectin-1α的MDCK细胞（Nectin-1-MDCK细胞）中，基于E-钙黏蛋白的细胞间黏附形成减少。在仅稳定表达外源性E-钙黏蛋白的L细胞（EL细胞）和野生型MDCK细胞中也观察到了Nef-3的这种拮抗作用。相反，包被有Nef-3的微珠首先在Nectin-1-EL和Nectin-1-MDCK细胞的珠-细胞接触位点募集Nectin-afadin复合物，然后募集E-钙黏蛋白-连环蛋白复合物。
这些结果表明，Nectin对于将E-钙黏蛋白募集到基于Nectin的细胞间黏附位点是必要且充分的，并且参与了基于E-钙黏蛋白的细胞间AJs的形成。
