MAPPING IN-VIVO ASSOCIATIONS OF CYTOPLASMIC PROTEINS WITH INTEGRIN BETA-1 CYTOPLASMIC DOMAIN MUTANTS
MAPPING IN-VIVO ASSOCIATIONS OF CYTOPLASMIC PROTEINS WITH INTEGRIN BETA-1 CYTOPLASMIC DOMAIN MUTANTS
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DOI：
10.1091/mbc.6.2.151
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发表时间：
1995-02-01
影响因子：
3.3
通讯作者：
SCHWARTZ, MA
中科院分区：
文献类型：
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作者：
LEWIS, JM;SCHWARTZ, MA
整合素促进粘着斑的形成并通过细胞质蛋白如talin、α-辅肌动蛋白和粘着斑激酶（FAK）触发细胞内信号传导途径。在体外试验中，β 1整联蛋白亚基已显示结合talin和α-辅肌动蛋白，并且这些蛋白质可将整联蛋白直接或通过与其他蛋白质如粘着斑蛋白的连接而连接到肌动蛋白细胞骨架。然而，目前尚不清楚这些关联中的哪些是体内形成局灶性接触所必需的，或者β 1整联蛋白的哪些区域在体内与特定的细胞骨架蛋白结合。我们已经开发了一种体内试验来解决这些问题。用抗鸡β 1抗体包被微珠，以选择性地聚集在培养的小鼠成纤维细胞中表达的鸡β 1整联蛋白。通过免疫荧光法评估胞质结构域突变体β 1整合素诱导蛋白质共定位的能力，并与野生型整合素进行比较。正如预期的那样，缺乏整个胞质结构域的突变体β 1诱导talin、α-辅肌动蛋白、F-肌动蛋白、黏着斑蛋白和FAK共定位的能力降低。发现β 1整联蛋白共定位talin和FAK的能力需要β 1的C-末端附近的序列。发现β 1共定位α-辅肌动蛋白所需的区域位于不同的序列中，距离β 1的C-末端有几个氨基酸。从C-末端删除13个残基阻断了talin、FAK和肌动蛋白的共定位，但不阻断α-辅肌动蛋白。因此，α-辅肌动蛋白与成簇整合素的结合不足以诱导F-肌动蛋白的共定位。
Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.