Massari Silvia, Perego Carla, Padovano Valeria, D'Amico Anna, Raimondi Andrea, Francolini Maura, Pietrini Grazia
Department of Medical Pharmacology, Institute of Neuroscience-Consiglio Nazionale delle Richerche, University of Milan, 20129 Milan, Italy.
Traffic. 2009 Feb;10(2):246-57. doi: 10.1111/j.1600-0854.2008.00854.x. Epub 2008 Nov 11.
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 (DeltaL27 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 (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). 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.
在本研究中，我们检测了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）组装过程中对支架蛋白LIN7蛋白质 - 蛋白质相互作用的作用，发现缺乏L27结构域的LIN7突变体（DeltaL27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发挥作用。LIN7的缺失并未改变L27结构域的PALS1（与LIN7相关的蛋白质）伴侣的定位，但阻止了LIN7的PDZ结构域的伴侣胰岛素受体底物p53（IRSp53）的TJ定位。通过降低LIN7的表达（LIN7小发夹RNA实验）以及表达缺失其PDZ相互作用基序的IRSp53（IRSp53Delta5）或与LIN7的L27结构域融合的IRSp53（L27 - IRSp53Delta5），进一步证明了LIN7的L27和PDZ结构域在IRSp53定位于TJ中的功能。LIN7和IRSp53定位于TJ减少的细胞系在TJ组装和囊肿极化过程中表现出缺陷，并且未能激活Rac1，Rac1是Rho鸟苷三磷酸酶家族的成员，在肌动蛋白组织和顶基极性的定向中起关键作用。因此，这些数据表明LIN7 - IRSp53相互作用在上皮细胞功能性TJ组装和表面极化过程中发挥作用。
