LIN7 Mediates the Recruitment of IRSp53 to Tight Junctions
LIN7 Mediates the Recruitment of IRSp53 to Tight Junctions
复制标题
DOI：
10.1111/j.1600-0854.2008.00854.x
复制
发表时间：
2009-02-01
期刊：
影响因子：
4.5
通讯作者：
Pietrini, Grazia
中科院分区：
文献类型：
--
作者：
Massari, Silvia;Perego, Carla;Pietrini, Grazia
在本研究中，我们研究了L27 [（LIN2-LIN7）结构域]和PDZ结构域（先前在PSD95-DlgA-ZO-1中发现的结构域）在Madin-Darby犬肾（MDCK）细胞紧密连接（TJ）组装中支架蛋白LIN7蛋白-蛋白相互作用中的作用，发现缺乏L27结构域的LIN7突变体（Delta L27突变体）的稳定表达通过抑制内源性LIN7的TJ定位而作为显性干扰蛋白。LIN7的缺失没有改变L27结构域的PALS1（与LIN7相关的蛋白）伴侣的定位，但阻止了胰岛素受体底物p53 （IRSp53）的TJ定位，IRSp53是LIN7的PDZ结构域的伴侣。通过降低LIN7的表达（LIN7小发卡RNA实验）和表达删除PDZ相互作用基序（IRSp53 Delta 5）或融合到LIN7的L27结构域（L27-IRSp53 Delta 5），进一步证明了LIN7的L27和PDZ结构域在IRSp53定位到TJs中的功能。LIN7和IRSp53在TJs上定位减少的细胞系在TJs组装和囊肿极化过程中出现缺陷，并且无法激活Rho鸟苷三磷酸酶家族成员Rac1， Rac1对肌动蛋白的组织和顶基极性的定向至关重要。因此，这些数据表明，LIN7-IRSp53结合在上皮细胞的功能性TJs组装和表面极化中起作用。
In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (Delta L27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53 Delta 5) or fused to the L27 domain of LIN7 (L27-IRSp53 Delta 5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.