Department of Molecular Biology, University of Geneva, 4 Boulevard d'Yvoy, 1205 Geneva, Switzerland.
J Biol Chem. 2012 Apr 13;287(16):13159-69. doi: 10.1074/jbc.M111.315622. Epub 2012 Feb 7.
Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.
副肌球蛋白是脊椎动物上皮紧密连接和黏附连接的一种 150-160kDa 的细胞质蛋白，包含球状头部、卷曲螺旋杆和球状尾部结构域。与同源紧密连接蛋白嵴蛋白不同，副肌球蛋白参与连接组装的调控，并与肌动蛋白丝一起定位于连接外区域。在此，我们通过在上皮 Madin-Darby 犬肾 (MDCK) 细胞中诱导过表达外源性蛋白以及在 Rat1 成纤维细胞和 MDCK 细胞中表达突变和嵌合构建体，分析副肌球蛋白结构域的作用以及头部和杆状结构域内的特定区域在副肌球蛋白功能和定位中的作用。副肌球蛋白的 rod+tail 结构域的过表达扰乱了钙开关诱导的紧密连接屏障的形成和 Rac1 的激活，表明副肌球蛋白对连接组装的调控是通过这些结构域介导的。相反，仅包含头部结构域的构建体可靶向 MDCK 细胞和 Rat1 成纤维细胞中的连接。此外，在 Rat1 成纤维细胞和 MDCK 细胞中表达嵌合嵴蛋白和副肌球蛋白构建体，确定了副肌球蛋白头部和杆状结构域内的特定序列分别是靶向肌动蛋白丝和调控连接组装的关键。总之，我们鉴定了副肌球蛋白的功能重要结构域，使其区别于嵴蛋白。
