RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells
RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells
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DOI：
10.1042/0264-6021:3590285
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发表时间：
2001-10-15
影响因子：
4.1
通讯作者：
Wilson-Delfosse, AL
中科院分区：
文献类型：
--
作者：
Gibson, RM;Wilson-Delfosse, AL
我们已经确定了人类G蛋白Cdc42Hs，R66E的突变体，不能形成一个可检测的复合物与GDP解离抑制剂RhoGDI在无细胞系统或在完整的细胞。该点突变体被异戊烯化，结合鸟嘌呤核苷酸，并以与野生型Cdc42H无区别的方式与GTP酶激活蛋白相互作用。免疫荧光定位研究表明，这种RhoGDI结合缺陷型突变体主要存在于高尔基体中，其染色模式与野生型蛋白相似。然而，不像野生型Cdc42Hs，这是分布在微粒体膜和胞质组分，研究使用差速离心表明，异戊二烯化R66E Cdc42Hs被发现专门与脂质双层。此外，而RhoGDI的过表达导致野生型Cdc42Hs从高尔基体到胞质溶胶的明显易位，相同的RhoGDI过表达条件不改变R66E突变体的高尔基定位。此外，这种RhoGDI结合缺陷突变体的Cdc42Hs的过表达似乎激活肌动蛋白细胞骨架和丝状伪足形成在成纤维细胞中的重新分配的方式与野生型蛋白质难以区分。两者合计，这些结果表明，Cdc42Hs与RhoGDI的相互作用是不必要的适当的膜靶向新生的异戊二烯化Cdc42Hs在哺乳动物细胞中，也不是这种相互作用的机制，Cdc42Hs激活丝状伪足形成的重要组成部分。然而，它似乎重新分配Cdc42Hs的胞质隔室是绝对依赖于RhoGDI的相互作用。
We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction.