Fukuhara Atsunori, Shimizu Kazuya, Kawakatsu Tomomi, Fukuhara Tatsuro, Takai Yoshimi
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
J Biol Chem. 2003 Dec 19;278(51):51885-93. doi: 10.1074/jbc.M308015200. Epub 2003 Oct 6.
Nectins, Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, trans-interact and form cell-cell adhesion, which increases the velocities of the formation of the E-cadherin-based adherens junctions (AJs) and the claudin-based tight junctions (TJs) in Madin-Darby canine kidney (MDCK) cells. The trans-interactions of nectins furthermore induce activation of Cdc42 and Rac small G proteins, but the roles of these small G proteins activated in this way remain unknown. We examined here the role and the mode of action of Cdc42 in the organization of AJs and TJs in MDCK cells. We first made the NWASP-Cdc42 and Rac interactive binding (CRIB) domain, an inhibitor of activated Cdc42, fused to the Ki-Ras CAAX motif (NWASP-CRIB-CAAX; where A is aliphatic amino acid), which was targeted to the cell-cell adhesion sites. We then found that overexpression of NWASP-CRIB-CAAX reduced the velocities of the formation of AJs and TJs. Conversely, overexpression of a constitutively active mutant of Cdc42 (V12Cdc42) increased their velocities, and the inhibitory effect of NWASP-CRIB-CAAX was suppressed by co-expression with V12Cdc42. The inhibitory effect of NWASP-CRIB-CAAX on the formation of AJs and TJs was suppressed by co-expression of nectin-1 of which trans-interaction activated endogenous Cdc42. Moreover, the formation of the claudin-based TJs required a greater amount of activated Cdc42 than that of the E-cadherin-based AJs. These results indicate that the Cdc42 activated by the trans-interactions of nectins is involved in the organization of AJs and TJs in different mechanisms in MDCK cells.
NECTIN蛋白是一种不依赖钙离子的免疫球蛋白样细胞间黏附分子，通过反式相互作用形成细胞间黏附，从而提高了犬肾上皮细胞（MDCK）中基于E-钙黏蛋白的黏附连接（AJs）和基于闭合蛋白的紧密连接（TJs）的形成速度。NECTIN蛋白的反式相互作用还能诱导Cdc42和Rac小G蛋白的激活，但以这种方式激活的这些小G蛋白的作用尚不清楚。我们在此研究了Cdc42在MDCK细胞中AJs和TJs组织中的作用及作用方式。我们首先构建了与Ki-Ras CAAX基序融合的NWASP-Cdc42和Rac相互作用结合（CRIB）结构域，即活化Cdc42的抑制剂（NWASP-CRIB-CAAX；其中A为脂肪族氨基酸），其靶向细胞间黏附位点。然后我们发现，NWASP-CRIB-CAAX的过表达降低了AJs和TJs的形成速度。相反，Cdc42组成型活性突变体（V12Cdc42）的过表达提高了它们的形成速度，并且与V12Cdc42共表达可抑制NWASP-CRIB-CAAX的抑制作用。与反式相互作用激活内源性Cdc42的NECTIN-1共表达可抑制NWASP-CRIB-CAAX对AJs和TJs形成的抑制作用。此外，基于闭合蛋白的TJs的形成比基于E-钙黏蛋白的AJs需要更多的活化Cdc42。这些结果表明，NECTIN蛋白反式相互作用激活的Cdc42通过不同机制参与MDCK细胞中AJs和TJs的组织形成。
