Single amino acid substitutions in conserved extracellular domains of E-cadherin differ in their functional consequences
Single amino acid substitutions in conserved extracellular domains of E-cadherin differ in their functional consequences
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DOI：
10.1006/jmbi.2001.5143
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发表时间：
2001-11-30
影响因子：
5.6
通讯作者：
Becker, KF
中科院分区：
文献类型：
--
作者：
Handschuh, G;Luber, B;Becker, KF
依赖钙的亲同性细胞粘附分子e -钙粘蛋白通常连接上皮细胞。成熟跨膜蛋白的胞外部分由五个同源结构域组成。连接这些结构域的四个序列包含结构氨基酸基序DXXD，该基序被认为参与钙的直接结合。在胃癌患者中，影响第二和第三结构域之间这一基序的突变是常见的。为了确定类似序列改变在其位置方面的功能意义，我们根据在胃癌中发现的突变（D370A）分析了将每个连接区域的DXXD基序改变为DXXA的单氨基酸取代。改变编码DQND、DVLD和DVND的cDNA序列（分别为D257A、D479A、D590A），并在E-cadherin阴性的MDA-MB-435S乳腺癌细胞中稳定表达。我们发现D257A和D370A突变导致蛋白定位异常，肌动蛋白细胞骨架改变，亲同型细胞粘附明显降低，细胞形态改变。出乎意料的是，肿瘤相关的D370A突变而不是D257A突变诱导细胞运动性增加。D479A突变只有轻微的功能影响，而表达D590A突变的细胞与表达野生型分子的细胞没有区别。虽然假定的钙结合基序DXXD位于E-cadherin细胞外部分的重复位置，但我们的研究结果表明，它具有不同的功能，取决于位置。值得注意的是，肿瘤细胞在最关键的区域选择突变，导致功能丧失（细胞粘附性降低）和功能获得（细胞运动性增加）。由于多个DXXD基序通常存在于其他钙粘蛋白中，因此我们的结构-功能研究通常与该基因家族相关。(C) 2001学术出版社。
The calcium-dependent homophilic cell adhesion molecule E-cadherin typically connects epithelial cells. The extracellular portion of the mature transmembrane protein consists of five homologous domains. The four sequences linking these domains contain the structural amino acid motif DXXD that is thought to be involved in direct calcium binding. In gastric cancer patients mutations affecting this motif between the second and third domain are frequently seen. In order to determine the functional significance of similar sequence alterations with regard to their location, we analyzed single amino acid substitutions changing the DXXD motif to DXXA in each linker region according to a mutation found in gastric cancer (D370A). The cDNA sequences coding for DQND, DVLD and DVND were changed (D257A, D479A, D590A, respectively) and stably expressed in E-cadherin negative MDA-MB-435S mammary carcinoma cells. We found that the D257A and D370A mutations result in abnormal protein localization, changes in the actin cytoskeleton, markedly reduced homophilic cell adhesion, and altered cell morphology. Unexpectedly, the tumor-associated D370A mutation but not the D257A mutation induced increased cell motility. The D479A mutation only had slight functional consequences whereas cells expressing the D590A mutant did not differ from cells expressing the wild-type molecule. Although the putative calcium binding motif DXXD is located at repetitive positions in the extracellular portion of E-cadherin, our results indicate that it has different functions depending on the location. Remarkably, tumor cells select for mutations in the most critical domains resulting both in loss of function (decreased cell adhesion) and in gain of function (increased cell motility). Since multiple DXXD motifs are typically seen in other cadherins, our structure-function study is relevant for this gene family in general. (C) 2001 Academic Press.