Actions of the Sodium-Hydrogen Exchanger Subtype, NHE1

描述(由申请人提供)：这项建议的总体目标是在分子水平上确定细胞内pH的变化如何调节细胞迁移。在前两个资金周期中，我们证实了细胞定向迁移需要通过Na-H交换器的H+外流。我们发现NHE1被锚定在肌动蛋白细丝上，肌动蛋白细丝定位于膜突起的远端边缘。我们发现NHE1在哺乳动物成纤维细胞和Dictyostelius细胞中的前沿H+外流是细胞迁移的三个阶段所必需的：极性、驱动膜突起的肌动蛋白细丝组装和细胞-底物黏附重塑。我们还开始研究pH传感器，也就是具有活性或配体结合亲和力的蛋白质，它们受pH的生理变化调节。细胞内pH传感器被预测为介导NHE1依赖的细胞迁移，通过计算模型、核磁共振和功能研究进行了检验。目前的提案调查了一种假设，即pH传感器在调节细胞迁移中发挥关键作用。我们的研究架起了细胞生物学和结构生物学的桥梁，在分子水平上确定了pH传感器在细胞极性、肌动蛋白依赖的膜突起和细胞黏附中的调节和功能。在目标1中，我们确定了成纤维细胞极性所需的NHE1和CDC42之间的正反馈环路的组件。我们将通过测试以下预测来询问NHE1如何刺激CDc42的活性，即介导NHE1依赖的CDc42激活的GEF是pH传感器，其pH依赖的PI(4，5)P2结合受组氨酸开关调节。我们还将通过测试预测，即增加NHE1的磷酸化是CDC42-GTP激活NHE1和细胞极性所必需的，来询问CDC42-GTP如何刺激NHE1的活性。在目标2中，我们确定了调节肌动蛋白细丝组装成膜突起的两相动力学的机制。我们将通过测试pH变化调节Aip1结构并与Cofilin结合来提供净肌动蛋白细丝组装和细胞迁移的预测，来探讨Cofilin和肌动蛋白相互作用蛋白1(Aip1)在NHE1依赖的肌动蛋白动力学中的作用。Arp2/3复合体Arp2亚单位的磷酸化是肌动蛋白细丝成核所必需的，这一新发现为我们提供了研究pArp2如何调节肌动蛋白动力学以响应迁移提示的理论基础。我们将通过生化分析和活细胞的高分辨率成像来测试Arp2的调节的磷酸化是否在运动的Dictyostelials细胞和哺乳动物成纤维细胞的肌动蛋白动力学和膜突起中具有不同的功能。在目标3中，我们确定了NHE1调节的焦点黏附蛋白在重塑细胞-基质黏附和成纤维细胞定向迁移中的作用。我们将通过测试FAK FERM结构域的组氨酸残基质子化立体地抑制自动磷酸化的预测来询问FAK的自动磷酸化是如何依赖于NHE1的。在细胞中的研究将测试突变的pH不敏感的FAK在焦点黏附动力学和细胞迁移中的功能。我们还将通过使用具有1-肌动蛋白结合位点突变的NHE来研究1-肌动蛋白与NHE1结合的功能意义。 公共卫生相关性：细胞迁移在许多生理过程中发挥关键作用，包括发育、轴突引导、免疫反应和伤口愈合，以及在病理学中，包括动脉粥样硬化和肿瘤细胞的转移和侵袭。由于细胞内pH是细胞定向迁移的进化保守的调节因子，在分子水平上识别pH调控的细胞迁移将有助于我们理解迁移阶段是如何被控制和整合的，以及如何被抑制以限制病理过程。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to determine at the molecular level how changes in intracellular pH regulate cell migration. In the previous two funding cycles we established that H+ efflux by the Na-H exchanger is necessary for directed cell migration. We found that NHE1 is anchored to actin filaments, which localizes NHE1 at the distal margin of membrane protrusions. We showed a leading-edge H+ efflux by NHE1 in mammalian fibroblasts and in Dictyostelium cells is necessary for three stages in cell migration: polarity, actin filament assembly driving membrane protrusion, and cell-substrate adhesion remodeling. We also began studying pH sensors, or proteins with activities or ligand-binding affinities that are regulated by physiological changes in pH. Intracellular pH sensors predicted to mediate NHE1-dependent cell migration were examined by computational modeling, NMR, and functional studies. The current proposal investigates the hypothesis that pH sensors play critical roles in regulating cell migration. Our studies bridge cell biology and structural biology to determine at the molecular level the regulation and function of pH sensors in cell polarity, actin-dependent membrane protrusion, and cell adhesion. In Aim 1 we identify components of the positive feedback loop between NHE1 and Cdc42 required for fibroblast cell polarity. We will ask how NHE1 stimulates Cdc42 activity by testing the prediction that GEFs mediating NHE1-dependent activation of Cdc42 are pH sensors with pH- dependent PI(4,5)P2 binding regulated by a histidine switch. We also will ask how Cdc42-GTP stimulates NHE1 activity by testing the prediction that increased phosphorylation of NHE1 is necessary for its activation by Cdc42-GTP and for cell polarity. In Aim 2 we identify mechanisms regulating the biphasic kinetics of actin filament assembly for membrane protrusion. We will ask how cofilin and actin-interacting protein 1 (Aip1) function in NHE1-dependent actin dynamics by testing the prediction that changes in pH regulate Aip1 structure and binding to cofilin to confer net actin filament assembly and cell migration. New findings that phosphorylation of the Arp2 subunit of the Arp2/3 complex is necessary for nucleating actin filaments provides the rationale to ask how pArp2 regulates actin kinetics in response to migratory cues. We will test whether regulated phosphorylation of Arp2 has distinct functions in actin dynamics and membrane protrusion in motile Dictyostelium cells and mammalian fibroblasts by using biochemical assays and high resolution imaging of live cells. In Aim 3 we determine the role of NHE1-regulated focal adhesion proteins in remodeling cell-substrate adhesions and in directed migration of fibroblasts. We will ask how autophosphorylation of FAK is NHE1- dependent by testing the prediction that protonation of histidine residues in the FERM domain of FAK sterically inhibits autophosphorylation. Studies in cells will test the function of a mutant pH-insensitive FAK in focal adhesion dynamics and cell migration. We also will ask the functional significance of 1-actinin binding to NHE1 by using NHE with mutations in the 1-actinin binding site. PUBLIC HEALTH RELEVANCE: Cell migration plays a critical role in a number of physiological processes, including development, axonal guidance, immune responses, and wound healing, and in pathologies, including atherosclerosis and the metastasis and invasion of tumor cells. Because intracellular pH is an evolutionarily-conserved regulator of directed cell migration, identifying pH-regulated cell migration at the molecular level will advance our understanding of how migratory stages are controlled and integrated and can be inhibited to restrict pathologies.