Fukuhara Atsunori, Irie Kenji, Nakanishi Hiroyuki, Takekuni Kyoji, Kawakatsu Tomomi, Ikeda Wataru, Yamada Akio, Katata Tatsuo, Honda Tomoyuki, Sato Tatsuhiro, Shimizu Kazuya, Ozaki Harunobu, Horiuchi Hisanori, Kita Toru, Takai Yoshimi
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
Oncogene. 2002 Oct 31;21(50):7642-55. doi: 10.1038/sj.onc.1205875.
Junctional adhesion molecule (JAM) is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which localizes at tight junctions (TJs). Claudin is a key cell-cell adhesion molecule that forms TJ strands at TJs. JAM is associated with claudin through their cytoplasmic tail-binding protein, ZO-1. JAM is furthermore associated with Par-3, a cell polarity protein which forms a ternary complex with Par-6 and atypical protein kinase C. Nectin is another Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which localizes at adherens junctions (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. We show here that nectin is furthermore involved in the localization of JAM at TJs. During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, JAM was recruited to the nectin-based cell-cell adhesion sites. This recruitment of JAM was inhibited by nectin inhibitors, which inhibited the trans-interaction of nectin. Microbeads coated with the extracellular fragment of nectin, that interacted with cellular nectin, also recruited JAM to the bead-MDCK cell contact sites. Furthermore, when cadherin-deficient L fibroblasts stably expressing both exogenous JAM and nectin (nectin-JAM-L cells) were co-cultured with L fibroblasts expressing only nectin (nectin-L cells), JAM was concentrated at the cell-cell adhesion sites between nectin-JAM-L and nectin-L cells without the trans-interaction of JAM. Analyses of the localization and immunoprecipitation of JAM revealed that it was associated with nectin through afadin and ZO-1. These results suggest that nectin has a role in the localization of JAM at TJs in the process of the formation of the junctional complex in epithelial cells.
连接黏附分子（JAM）是一种不依赖钙离子的免疫球蛋白样细胞间黏附分子，定位于紧密连接（TJ）处。闭合蛋白是一种关键的细胞间黏附分子，在紧密连接处形成紧密连接链。JAM通过其胞质尾结合蛋白ZO-1与闭合蛋白相关联。此外，JAM还与Par-3相关联，Par-3是一种细胞极性蛋白，它与Par-6和非典型蛋白激酶C形成三元复合物。NECTIN是另一种不依赖钙离子的免疫球蛋白样细胞间黏附分子，定位于黏着连接（AJ）处。NECTIN通过其各自的胞质尾结合蛋白afadin和连环蛋白与E-钙黏蛋白相关联，并与E-钙黏蛋白协同参与黏着连接的形成。我们在此表明，NECTIN还参与JAM在紧密连接处的定位。在Madin-Darby犬肾（MDCK）细胞中由黏着连接和紧密连接组成的连接复合物形成过程中，JAM被招募到基于NECTIN的细胞间黏附位点。这种JAM的招募被NECTIN抑制剂抑制，该抑制剂抑制了NECTIN的反式相互作用。包被有NECTIN细胞外片段的微珠与细胞内NECTIN相互作用，也将JAM招募到微珠-MDCK细胞接触位点。此外，当稳定表达外源性JAM和NECTIN的钙黏蛋白缺陷型L成纤维细胞（NECTIN-JAM-L细胞）与仅表达NECTIN的L成纤维细胞（NECTIN-L细胞）共培养时，JAM在NECTIN-JAM-L和NECTIN-L细胞之间的细胞间黏附位点处聚集，而无需JAM的反式相互作用。对JAM的定位和免疫沉淀分析表明，它通过afadin和ZO-1与NECTIN相关联。这些结果表明，NECTIN在上皮细胞连接复合物形成过程中对JAM在紧密连接处的定位起作用。
