Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition
Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition
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DOI：
10.1073/pnas.95.5.2515
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发表时间：
1998-03-03
影响因子：
11.1
通讯作者：
Frangos, JA
中科院分区：
文献类型：
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作者：
Gudi, S;Nolan, JP;Frangos, JA
由应变、压力和流体剪切应力引起的机械力由细胞通过与细胞内信号传导途径偶联的未鉴定的机械感受器感知。在血管内皮细胞中，流体剪切应力通过涉及异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白）的途径通过未知的分子机制转导。在本研究中，我们研究了活化纯化的G蛋白重组成磷脂囊泡。将含有G蛋白的囊泡加载[γ-P-32]GTP，并在锥板粘度计中经受生理水平的流体剪切应力。测定稳态GTP水解作为G蛋白功能的指标。剪切应力（0-30达因/cm（2））以剂量依赖性方式激活G蛋白（0.48-4.6 pmol/min/pg蛋白）。脂质体含有溶血磷脂酰胆碱（30摩尔%）或苄醇（40 mM），增加双层流动性的条件下，表现出3至5倍的基础GTdR活性增强。相反，掺入胆固醇（24摩尔%）到脂质体减少G蛋白的激活剪切。这些结果表明，磷脂双层介导的剪切应力诱导的活化膜结合的G蛋白在蛋白受体的情况下，双层的物理性质调节这种反应的能力。
Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [gamma-P-32]GTP and subjected to physiological Levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0-30 dynes/cm(2)) activated G proteins in dose-dependent manner (0.48-4.6 pmol/min per pg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.