The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells
The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells
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DOI：
10.1016/j.yexcr.2005.04.015
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发表时间：
2005-08-01
影响因子：
3.7
通讯作者：
Guillemette, G
中科院分区：
文献类型：
--
作者：
Auger-Messier, M;Turgeon, ES;Guillemette, G
一个结构性活性的G蛋白偶联受体的表达有望触发从正常到适应性反应的各种细胞变化。我们报道了稳定表达血管紧张素11的组成性活性突变体N111G-AT(1)受体的融合HEK-293细胞自发地表现出戏剧性的形态变化和细胞骨架重组。相差显微镜显示，这些细胞形成致密的单层，而表达WT-AT(1)受体的细胞显示出巨大的细胞间隙和大量的丝状足细胞。共聚焦显微镜显示，在表达N111G-AT(1)受体的细胞的顶端和基底外侧表面，有一个复杂的聚合肌动蛋白网络。有趣的是，通过在反向激动剂EXP3174存在的情况下培养细胞来防止这些表型变化。在表达WT-AT(1)受体的血管紧张素Ⅱ刺激的细胞中，发现相似的形态重排和从头聚合的肌动蛋白结构。我们进一步表明，AT(1)受体诱导的细胞-细胞接触的形成不需要增加细胞内钙离子浓度或蛋白激酶C的活性。然而，Y-27632的预处理表明，在AT-1受体激活时，细胞-细胞接触的形成需要Rho-Kinase的活性。这些观察表明，结构性活性突变体N111G-AT(1)受体的表达对HEK-293细胞的形态和细胞骨架组织有显著影响，可能是通过参与Rho-Kinase活性的机制。(C)2005 Elsevier Inc.保留所有权利。
The expression of a constitutively active G protein-coupled receptor is expected to trigger diverse cellular changes ranging from normal to adaptive responses. We report that confluent HEK-293 cells stably expressing the constitutively active mutant N111G-AT(1) receptor for angiotensin 11 spontaneously exhibited dramatic morphological changes and cytoskeletal reorganization. Phase-contrast microscopy revealed that these cells formed a dense monolayer, whereas cells expressing the WT-AT(1) receptor displayed large intercellular spaces and numerous filopodia. Confocal microscopy revealed an elaborate web of polymerized actin at the apical and basolateral surfaces of cells expressing the N111G-AT(1) receptor. Interestingly, these phenotypic changes were prevented by culturing the cells in the presence of the inverse agonist EXP3174. Similar morphologic rearrangements and de novo polymerized actin structures were found in Ang II-stimulated cells expressing the WT-AT(1) receptor. We further showed that AT(1) receptor-induced cell-cell contact formation did not require an increase in intracellular Ca2+ concentration or the activity of protein kinase C. However, pretreatment with Y-27632 revealed that Rho-kinase activity was required for cell-cell contact formation upon AT, receptor activation. These observations demonstrate that the expression of the constitutively active mutant N111G-AT(1) receptor had a significant impact on the morphology and cytoskeletal organization of HEK-293 cells, possibly via a mechanism involving the activity of Rho-kinase. (c) 2005 Elsevier Inc. All rights reserved.