Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane
Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane
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DOI：
10.1128/mcb.24.15.6799-6810.2004
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发表时间：
2004-08-01
影响因子：
5.3
通讯作者：
Hancock, JF
中科院分区：
文献类型：
--
作者：
Rotblat, B;Prior, IA;Hancock, JF
Ras蛋白微定位于质膜的不同微区对于信号传导特异性至关重要。在这里，我们研究了复杂的膜相互作用的H-ras与活细胞上的FRAP的组合，以测量膜亲和力和电子显微镜的完整质膜片的空间映射微区。我们发现，三个可分离的力量在质膜上的H-ras。脂质锚包含加工的CAAX基序和两个棕榈酸残基，产生一种吸引力，其提供与脂筏的高亲和力相互作用。相邻的高变连接域提供了第二个吸引力，但非筏质膜微区。与脂筏的脂质锚的吸引相互作用相反的是由N-末端催化结构域产生的排斥力，当H-ras被GTP负载时，该排斥力增加。这些观察结果直接导致了一种新的机制，解释了如何H-ras的横向隔离调节激活状态：GTP负载降低H-ras的脂筏亲和力，并允许高变连接域的目标nonraft微域，H-ras信号的主要网站。
The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.