Mechanisms of Receptor Regulated Na+-H+ Exchange

受体调节 Na -H 交换的机制

• 批准号: 7889280
• 负责人: DIANE L BARBER
• 金额: $ 7.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7889280
• 关键词: Actin-Binding Protein; Actins; Affinity; Affinity Chromatography; Binding; C-terminal; Cell Proliferation; Cell Shape; Cell Volumes; Cells; Complex; Cytoplasmic Tail; Distal; Fibroblasts; Focal Adhesions; Funding; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Integrins; Ions; Link; Mass Spectrum Analysis; Mediating; Membrane; Microfilaments; Multiprotein Complexes; Mutation; Myosin Light Chains; NHE1; Neoplastic Cell Transformation; Normal Cell; Null Lymphocytes; Pathway interactions; Phosphorylation; Phosphotransferases; Protein Binding; Proteins; Proteomics; Regulation; Rho-associated kinase; Role; Signal Transduction; Signaling Molecule; Site; Sum; Testing; extracellular; ezrin; growth factor-activatable Na-H exchanger NHE-1; moesin; mutant; pH Homeostasis; radixin protein; receptor; scaffold; tumor progression

DESCRIPTION (provided by applicant): The ubiquitously expressed Na-H exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral exchange of extracellular Na + for intracellular H+. During the past funding period an ancillary function of NHEI as an anchor for actin filaments was identified. Actin anchoring by NHE1 is mediated by direct binding of ERM (ezrin, radixin, moesin) actin-binding proteins, and in fibroblasts is necessary to retain the predominant localization of NHEI at the distal margin of lamellipodia, normal cell shape, and the assembly of cortical actin filaments. The objective of the current proposal is to establish an additional ancillary function of NHEI as a scaffolding platform for assembling signaling complexes and promoting signal relay. The C-terminal cytoplasmic domain of NHE1 binds directly at least 10 functionally distinct signaling molecules that coordinately regulate NHE1 activity, restrict the localization of NHE1 in specialized membrane domains, and as now proposed, may transmit signals to diverse effector pathways. Focusing on the role of NHE1 in fibroblasts, this proposal investigates the hypothesis that NHE1 acts as a spatially-restricted scaffold to assemble signaling complexes and to promote signal relay. Studies in Aim 1 will determine how NHE1 scaffolding and localization are integrated by asking how scaffolding by NHE1 determines its localization and in turn how the localization of NHE1 in lamellipodia determines its binding to identified interacting proteins. Studies in Aim 2 will test the role of an NHE1 scaffold in the assembly of signaling complexes and in signal relay. Distinct signaling units, each having a component that binds directly to NHE1, will be investigated by asking whether binding to NHE1, the localization of NHE1 in lamellipodia, or ion translocation by NHE1 is necessary for the assembly and signal relay of the unit. Representative signaling units include phosphorylation of ezrin and Arp2/3 by the Ste20-1ike kinase NIK, ezrin binding to the GTPases Rac and Cdc42, and phosphorylation of myosin light chain by the Rho kinase ROCK. Studies in Aim 3 will define the complexity of an NHE1 scaffold by using a proteomics approach to determine the dynamic composition of an NHE1 scaffold ensemble and then will ask whether the profile of associated proteins is different when NHE 1 localization or ion translocation is impaired. Established roles for NHEI in intracellular pH homeostasis, cell proliferation, neoplastic transformation, and tumor progression underscore the importance of understanding its structural and functional regulation of diverse signaling networks.

描述（由申请人提供）：普遍表达的 Na-H 交换剂 NHE1 通过催化细胞外 Na + 与细胞内 H + 的电中性交换，在细胞内 pH 和细胞体积稳态中具有确定的功能。在过去的资助期间，NHEI 作为肌动蛋白丝锚的辅助功能被确定。 NHE1 锚定的肌动蛋白是通过 ERM（埃兹蛋白、根蛋白、模蛋白）肌动蛋白结合蛋白的直接结合介导的，并且在成纤维细胞中，对于保留 NHEI 在板状伪足远端边缘的主要定位、正常细胞形状和组装皮质肌动蛋白丝。当前提案的目标是建立 NHEI 的额外辅助功能，作为组装信号复合物和促进信号传递的脚手架平台。 NHE1 的 C 端胞质结构域直接结合至少 10 个功能不同的信号分子，这些信号分子协调调节 NHE1 活性，限制 NHE1 在专门的膜结构域中的定位，并且正如现在所提出的，可以将信号传递到不同的效应通路。该提案重点关注 NHE1 在成纤维细胞中的作用，研究了 NHE1 作为空间限制支架来组装信号复合物并促进信号传递的假设。目标 1 中的研究将通过询问 NHE1 的支架如何确定其定位以及 NHE1 在板状伪足中的定位如何确定其与已识别的相互作用蛋白的结合来确定 NHE1 支架和定位如何整合。目标 2 的研究将测试 NHE1 支架在信号复合物组装和信号传递中的作用。通过询问与 NHE1 的结合、NHE1 在片状伪足中的定位或 NHE1 的离子易位对于该单元的组装和信号传递是否是必需的，将研究不同的信号单元，每个单元都有一个直接与 NHE1 结合的组件。代表性的信号传导单元包括由 Ste20-1ike 激酶 NIK 磷酸化埃兹蛋白和 Arp2/3、与 GTPases Rac 和 Cdc42 结合的埃兹蛋白以及由 Rho 激酶 ROCK 磷酸化肌球蛋白轻链。目标 3 中的研究将通过使用蛋白质组学方法确定 NHE1 支架整体的动态组成来定义 NHE1 支架的复杂性，然后将询问当 NHE 1 定位或离子易位受损时相关蛋白质的特征是否不同。 NHEI 在细胞内 pH 稳态、细胞增殖、肿瘤转化和肿瘤进展中的既定作用强调了了解其对不同信号网络的结构和功能调节的重要性。