Actions of the Sodium-Hydrogen Exchanger Subtype, NHE1

描述（由申请人提供）：本提案的总体目标是在分子水平上确定细胞内pH值的变化如何调节细胞迁移。在前两个资助周期中，我们确定了Na-H交换器的H+流出对于定向细胞迁移是必要的。我们发现NHE1被锚定在肌动蛋白丝上，这使得NHE1定位在膜突起的远端边缘。我们发现，NHE1在哺乳动物成纤维细胞和盘形骨细胞中的前沿H+外排对于细胞迁移的三个阶段是必要的：极性，肌动蛋白丝组装驱动膜突出，细胞-底物粘附重塑。我们还开始研究pH传感器，或受pH生理变化调节的具有活性或配体结合亲和力的蛋白质。通过计算模型、核磁共振和功能研究，预测介导nhe1依赖性细胞迁移的细胞内pH传感器。目前的提议调查了pH传感器在调节细胞迁移中起关键作用的假设。我们的研究将细胞生物学和结构生物学联系起来，在分子水平上确定pH传感器在细胞极性、肌动蛋白依赖的膜突起和细胞粘附中的调节和功能。在Aim 1中，我们确定了成纤维细胞极性所需的NHE1和Cdc42之间的正反馈回路的组成部分。我们将通过验证介导NHE1依赖性Cdc42激活的GEFs是pH传感器，其pH依赖性PI(4,5)P2结合由组氨酸开关调节，从而验证NHE1如何刺激Cdc42活性。我们还将通过测试预测NHE1磷酸化的增加是Cdc42-GTP激活和细胞极性所必需的，来询问Cdc42-GTP是如何刺激NHE1活性的。在目标2中，我们确定了调节肌动蛋白丝组装双相动力学的机制。我们将通过测试预测pH变化调节Aip1结构并与cofilin结合从而赋予净肌动蛋白丝组装和细胞迁移，来探讨cofilin和肌动蛋白相互作用蛋白1 （Aip1）如何在nhe1依赖性肌动蛋白动力学中起作用。Arp2/3复合物的Arp2亚基的磷酸化对于肌动蛋白丝的成核是必要的，这一新发现为研究pArp2如何调节肌动蛋白动力学以响应迁移线索提供了理论依据。我们将通过生化分析和活细胞的高分辨率成像来测试Arp2的调控磷酸化是否在运动盘形骨细胞和哺乳动物成纤维细胞的肌动蛋白动力学和膜突出中具有不同的功能。在Aim 3中，我们确定了nhe1调控的局灶黏附蛋白在重塑细胞-底物黏附和成纤维细胞定向迁移中的作用。我们将通过测试FAK的FERM结构域中组氨酸残基的质子化会在空间上抑制自磷酸化的预测，来询问FAK的自磷酸化是如何依赖于NHE1的。细胞研究将测试突变ph不敏感FAK在局灶黏附动力学和细胞迁移中的功能。我们还将通过使用1-actin结合位点突变的NHE来探讨1-actin与NHE1结合的功能意义。

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.