Ras membrane orientation and nanodomain localization generate isoform diversity
Ras membrane orientation and nanodomain localization generate isoform diversity
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DOI：
10.1073/pnas.0903907107
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发表时间：
2010-01-19
影响因子：
11.1
通讯作者：
Hancock, John F.
中科院分区：
文献类型：
--
作者：
Abankwa, Daniel;Gorfe, Alemayehu A.;Hancock, John F.
在Ras GTP酶中编码功能多样性的结构元件定义不明确。H - ras的G结构域相对于质膜平面的取向可被C - Raf的Ras结合结构域识别，这种取向与MAPK激活相关联。我们现在表明，另外两种蛋白质，磷脂酰肌醇 - 3 - 激酶 - α和结构上不相关的半乳糖凝集素 - 1，也能识别G结构域的取向。这些结果合理地解释了半乳糖凝集素 - 1在产生有活性的GTP - H - ras信号纳米簇中的作用。然而，对K - ras膜插入的分子动力学模拟以及对N - Ras、K - Ras和M - ras效应物相互作用的荧光寿命成像显微镜（FLIM） - 福斯特共振能量转移（FRET）成像表明，Ras G结构域存在两种活性过高、具有信号传导能力的取向。突变和功能分析确定了效应物结合与α4螺旋和C末端高变区的两亲性之间的明确关系，从而证实这些结构元件对Ras G结构域的取向起着关键的调节作用。最后，我们表明G结构域取向和纳米簇协同作用，在效应物相互作用方面产生Ras异构体特异性。
The structural elements encoding functional diversity among Ras GTPases are poorly defined. The orientation of the G domain of H-ras with respect to the plane of the plasma membrane is recognized by the Ras binding domain of C-Raf, coupling orientation to MAPK activation. We now show that two other proteins, phosphoinositide-3-kinase-alpha and the structurally unrelated galectin-1, also recognize G-domain orientation. These results rationalize the role of galectin-1 in generating active GTP-H-ras signaling nanoclusters. However, molecular dynamics simulations of K-ras membrane insertion and fluorescence lifetime imaging microscopy (FLIM)-Forster resonance energy transfer (FRET) imaging of the effector interactions of N-Ras, K-Ras, and M-ras suggest that there are two hyperactive, signaling-competent orientations of the Ras G domain. Mutational and functional analyses establish a clear relationship between effector binding and the amphilicities of helix alpha 4 and the C-terminal hypervariable region, thus confirming that these structural elements critically tune the orientation of the Ras G domain. Finally, we show that G-domain orientation and nanoclustering synergize to generate Ras isoform specificity with respect to effector interactions.