Actions of the Sodium-Hydrogen Exchanger Subtype, NHE1

钠氢交换子亚型 NHE1 的作用

基本信息

批准号：
8242039
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
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8242039
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 abstracting actin interacting protein 1 axonal guidance cell motility cofilin gene replacement membrane assembly migration mutant prevent protonation response sensor structural biology

项目摘要

PROJECT SUMMARY/ABSTRACT 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 ¿-actinin binding to NHE1 by using NHE1 with mutations in the ¿-actinin binding site.

项目摘要/摘要确定在分子水平的总体目标调节细胞迁移。对于定向的细胞迁移是必需的。 NHE1在膜突起的远端边缘。在细胞迁移的三个阶段中，必须使用哺乳动物的FIROBLASTS和dictyostelium细胞中的细胞：极性，肌动蛋白灯丝组件驱动膜原质和细胞覆盖重塑研究pH传感器或具有活性定制的活性或配体结合亲和力的蛋白质检验了pH。检测pH。被预测将介导NHE1依赖性细胞迁移的细胞内pH传感器的变化通过计算机建模，NMR和功能研究。 pH传感器在调节细胞迁移中起关键作用。要在分子水平确定pH传感器在细胞极性中的调节和功能膜突出和AIM 1中的细胞粘附。 NHE1和CDC42在FIROMBLAST细胞极性中需要询问NHE1如何刺激Cdc42活性通过测试介导NHE1依赖性激活Cdc42的GEFS的预测是具有pH-的pH传感器依赖性PI（4,5）P2由组氨酸开关定制。 NHE1活性通过测试预测，NHE1的增量磷酸化是为了激活通过CDC42-GTP和AIM 2中的细胞极性。膜突起的细丝组件。通过测试pH调节AIP1的预测，在NHE1依赖性肌动蛋白动力学中的功能结构并结合限量，以赋予净肌动蛋白丝和细胞迁移 ARP2/3复合物的ARP2亚基的磷酸化对于核素丝是必要的询问PARP2如何对迁移提示的肌动蛋白动力学的规则原理。 ARP2的调节phosporition在肌动蛋白动力学和膜上具有不同的功能使用生化测定和现场高分辨率成像，柱状细胞和哺乳动物成纤维细胞通过细胞在AIM 3中，我们确定NHE1型焦点粘附蛋白在重塑细胞底物中的作用在成纤维细胞的粘附和登记的迁移中。通过测试预测fak Ferm域中组氨酸属质的质子化依赖性抑制自噬的细胞研究将测试粘附动力学和细胞迁移。 - 肌动蛋白与NHE1结合通过使用NHE1与突变中的突变 - 肌动蛋白结合位点。