Actions of the Sodium-Hydrogen Exchanger Subtype, NHE1

钠氢交换子亚型 NHE1 的作用

• 批准号: 8050700
• 负责人: DIANE L BARBER
• 金额: $ 34.07万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050700
• 关键词: Actin-Binding Protein; Actinin; Actins; Adhesions; Affinity; Atherosclerosis; Attenuated; Automobile Driving; Binding; Binding Sites; Biochemical; Biological Assay; Catalytic Domain; Cell Adhesion; Cell Polarity; Cell Surface Extensions; Cells; Cellular biology; Chemotaxis; Complex; Computer Simulation; Cues; Development; Dictyostelium; Distal; Feedback; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesions; Funding; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Histidine; Image; Immune response; Kinetics; Life; Ligand Binding; Mammalian Cell; Mediating; Membrane; Microfilaments; Molecular; Mutation; NHE1; Neoplasm Metastasis; Null Lymphocytes; PTK2 gene; Pathology; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiological Processes; Play; Proteins; Regulation; Resolution; Role; Signal Transduction; Sodium-Hydrogen Antiporter; Staging; Structure; Talin; Testing; Tumor Cell Invasion; Wound Healing; actin interacting protein 1; axonal guidance; cell motility; cofilin; gene replacement; membrane assembly; migration; mutant; prevent; protonation; public health relevance; response; sensor; structural biology

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.

描述（由申请人提供）：该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 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 锚定在肌动蛋白丝上，将 NHE1 定位在膜突起的远端边缘。我们发现，在哺乳动物成纤维细胞和盘基网柄菌细胞中，NHE1 的前沿 H+ 流出对于细胞迁移的三个阶段是必需的：极性、驱动膜突出的肌动蛋白丝组装和细胞-基质粘附重塑。我们还开始研究 pH 传感器，即具有受 pH 生理变化调节的活性或配体结合亲和力的蛋白质。通过计算模型、NMR 和功能研究对预测介导 NHE1 依赖性细胞迁移的细胞内 pH 传感器进行了检查。目前的提案研究了 pH 传感器在调节细胞迁移中发挥关键作用的假设。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、肌动蛋白依赖性膜突出和细胞粘附中的调节和功能。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成部分。我们将通过测试以下预测来了解 NHE1 如何刺激 Cdc42 活性：介导 NHE1 依赖性 Cdc42 激活的 GEF 是 pH 传感器，具有由组氨酸开关调节的 pH 依赖性 PI(4,5)P2 结合。我们还将通过测试预测来询问 Cdc42-GTP 如何刺激 NHE1 活性，即 NHE1 磷酸化的增加对于 Cdc42-GTP 的激活和细胞极性是必需的。在目标 2 中，我们确定了调节膜突出肌动蛋白丝组装双相动力学的机制。我们将通过测试 pH 变化调节 Aip1 结构以及与肌动蛋白丝切蛋白结合以赋予净肌动蛋白丝组装和细胞迁移的预测，来了解肌动蛋白丝切蛋白和肌动蛋白相互作用蛋白 1 (Aip1) 在 NHE1 依赖性肌动蛋白动力学中如何发挥作用。新发现表明，Arp2/3 复合物的 Arp2 亚基的磷酸化对于肌动蛋白丝成核是必需的，这为探究 pArp2 如何调节肌动蛋白动力学以响应迁移线索提供了基础。我们将通过使用生化测定和活细胞的高分辨率成像来测试 Arp2 的调节磷酸化是否在运动盘基网柄菌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独特的功能。在目标 3 中，我们确定了 NHE1 调节的粘着斑蛋白在重塑细胞-基质粘附和成纤维细胞定向迁移中的作用。我们将通过测试 FAK FERM 结构域中组氨酸残基的质子化在空间上抑制自磷酸化的预测来询问 FAK 的自磷酸化如何是 NHE1 依赖性的。细胞研究将测试 pH 不敏感突变型 FAK 在粘着斑动力学和细胞迁移中的功能。我们还将通过使用 1-辅肌动蛋白结合位点发生突变的 NHE 来询问 1-辅肌动蛋白与 NHE1 结合的功能意义。 公共健康相关性：细胞迁移在许多生理过程中发挥着关键作用，包括发育、轴突引导、免疫反应和伤口愈合，以及在病理学中发挥着关键作用，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 值是定向细胞迁移的进化保守调节剂，因此在分子水平上识别 pH 调节的细胞迁移将促进我们对迁移阶段如何控制和整合以及如何被抑制以限制病理的理解。