Rosenberger Georg, Jantke Inka, Gal Andreas, Kutsche Kerstin
Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Butenfeld 42, 22529 Hamburg, Germany.
Hum Mol Genet. 2003 Jan 15;12(2):155-67. doi: 10.1093/hmg/ddg019.
Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton. It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity. Moreover, mutations in ARHGEF6 (also known as alphaPIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation. In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (beta-parvin or affixin) as a novel binding partner. The interaction was confirmed by co-immunoprecipitation and GST pull-down. We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin. In addition, interaction of ARHGEF6 to ARHGEF7 (betaPIX or Cool-1), a close homolog of ARHGEF6, was confirmed. In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 deltaaa56-83 and deltaaa396-776, abolished interaction of ARHGEF6 to PARVB. Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 deltaaa56-83 but did not occur with ARHGEF6 deltaaa396-776. These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding. In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7. PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell-substrate interaction through integrins. The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42.
Rho GTPase家族成员是关键的调节分子，可将表面受体与肌动蛋白细胞骨架的组织联系起来。现已明确，这些小GTP酶对神经元形态发生和连接也至关重要。此外，编码Rac1/Cdc42特异性鸟嘌呤核苷酸交换因子的ARHGEF6（也称为alphaPIX或Cool-2）中的突变与X连锁智力迟钝有关。为了深入了解ARHGEF6的生物学功能以及导致其激活的上游信号级联反应，我们在酵母双杂交筛选中使用ARHGEF6的全长编码区作为诱饵，并鉴定出PARVB（β-parvin或affixin）为一种新的结合伴侣。通过免疫共沉淀和GST下拉实验证实了这种相互作用。我们通过免疫荧光显示，在黏附于纤连蛋白的铺展良好且正在积极铺展的细胞中，ARHGEF6和PARVB共定位于细胞周边的片状伪足和褶皱处。此外，还证实了ARHGEF6与ARHGEF6的紧密同源物ARHGEF7（βPIX或Cool-1）之间的相互作用。在体内实验中，先前在X连锁非特异性智力迟钝患者中鉴定出的两个ARHGEF6突变体ARHGEF6 deltaaa56 - 83和deltaaa396 - 776，消除了ARHGEF6与PARVB之间的相互作用。ARHGEF6 deltaaa56 - 83不影响ARHGEF6与ARHGEF7之间的结合，但ARHGEF6 deltaaa396 - 776则不会发生这种结合。这些数据表明，N端的钙调蛋白同源（CH）结构域和C端的卷曲螺旋结构域对于ARHGEF6 - PARVB的结合都是必需的。相比之下，似乎只有卷曲螺旋结构域是ARHGEF6与ARHGEF7相互作用和异二聚化所必需的。已知PARVB与整合素连接激酶（ILK）相互作用，并通过整合素参与细胞 - 底物相互作用的早期阶段。PARVB作为ARHGEF6相互作用伴侣的鉴定以及ARHGEF6与ILK在铺展细胞中的共定位表明，ARHGEF6参与整合素介导的信号传导，导致GTP酶Rac1和/或Cdc42的激活。
