Mechanisms of Receptor Regulated Na+-H+ Exchange

受体调节 Na -H 交换的机制

• 批准号: 8320968
• 负责人: DIANE L BARBER
• 金额: $ 36.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320968
• 关键词: 1-Phosphatidylinositol 3-Kinase; 6-Phosphofructokinase; Acidosis; Address; Affect; Affinity; Animal Model; Apoptosis; Architecture; Attenuated; Binding; Biochemical; C-terminal; Carcinoma; Cardiac; Cell Cycle Progression; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cerebral Ischemia; Computer Simulation; Cytoplasmic Tail; Data; Dependence; Diabetes Mellitus; Disseminated Malignant Neoplasm; Drosophila eye; Drosophila genus; Enzymes; Funding; G2/M Transition; Genetic; Genetic Transcription; Glycolysis; Goals; Grant; Growth Factor; Hematologic Neoplasms; In Situ; In Vitro; Learning; Malignant Neoplasms; Mediating; Mediator of activation protein; Metabolic; Metabolism; Mitosis; Mitotic; Modeling; Molecular; NHE1; Necrosis; Neoplasm Metastasis; Normal Cell; Normal Range; Oncogene Activation; Oncogenes; Pathway interactions; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Play; Protein Biochemistry; Protein Dephosphorylation; Proteins; Regulation; Retinal; Role; S Phase; Signal Transduction; Solid Neoplasm; Structure; Testing; Therapeutic; Time; Tissues; Tumor Suppressor Proteins; base; cancer cell; cdc25 Phosphatase; cell motility; cell transformation; cyclin B1; design; driving force; innovation; macromolecular assembly; migration; mutant; overexpression; prevent; protein structure; receptor; scaffold; sensor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): Dynamic changes in intracellular pH (pHi) regulate a range of normal and pathological cell processes. Increased pHi promotes cell proliferation, differentiation, and migration, and decreased pHi induces apoptosis. Dysregulated pHi is thought to contribute to cancer progression, diabetes, and tissue damage after cardiac and cerebral ischemia. The long-term goals of this grant are to determine the regulation and function of pHi dynamics. The past four funding cycles focused on how H+ fluxes by the plasma membrane Na-H exchanger NHE1 are regulated and drive pHi dynamics. We also showed the functional significance of NHE1-dependent increases in pHi for cell proliferation, cell cycle progression, and cell migration. Despite the broad significance of pHi-dependent cell functions, we have limited understanding of how changes in pHi affect proteins and macromolecular assemblies. To address this limitation we recently began studying the structure and function of pH sensors, or proteins with activities or binding affinities that are sensitive to small physiological changes in pH. The current application applies what we collectively learned in the previous four funding cycles to determine the design principles and function of pH sensors regulating basic cell processes that are aberrant in cancer cells. Increased pHi is a hallmark of most cancers, regardless of the tissue origin or genetic background. This likely reflects a dependence on higher pHi for metabolic adaptation, increased proliferation, and metastasis. We will test the hypothesis that pH sensors controlling metabolism and cell cycle progression play critical roles in how NHE1 and pHi direct cell functions that are dysregulated in cancer. Our studies use an innovative comprehensive approach that bridges protein structure, protein biochemistry, and cell physiology. In Aim 1 we will determine how kinases controlling metabolism are regulated by NHE1. We will test predictions on how phosphoinositide 3-kinase is pH sensitive, based on our recent structural and biochemical findings that its p85 regulatory subunit is a pH sensor. We also will determine NHE1 regulation of phosphofructokinase 1 (PFK1), the first rate-limiting enzyme in glycolysis, which directly binds the C-terminal cytoplasmic domain of NHE1 and has extremely pH-sensitive activity. In Aim 2 we will determine how NHE1 promotes cell proliferation by focusing on our previous findings that H+ efflux by NHE1 times G2/M. We will test predictions on Wee1 as a putative pH sensor based on our new computational and biochemical data and on how cyclin B1 expression is attenuated in cells lacking NHE1 activity. In Aim 3 we will determine the role of NHE activity and dysregulated pHi in tumorigenesis using Drosophila models we generated. We will test predictions on how loss of Dnhe2 may be a synthetic lethal for transformed but not normal cells, and test rescue of phenotypes with mutant pH sensors generated in Aim 1 and 2. We also will ask whether over expression of Dnhe2 cooperates with oncogene activation or tumor suppressor deletion to induce metastatic cancer, and will use modifier screens to identify mediators of the Dnhe2 over expression phenotype.

描述（由申请人提供）：细胞内 pH (pHi) 的动态变化调节一系列正常和病理细胞过程。 pHi 升高促进细胞增殖、分化和迁移，pHi 降低则诱导细胞凋亡。 pHi 失调被认为会导致癌症进展、糖尿病以及心脑缺血后的组织损伤。此项资助的长期目标是确定 pHi 动力学的调节和功能。过去的四个资助周期重点关注质膜 Na-H 交换器 NHE1 的 H+ 通量如何受到调节并驱动 pHi 动态。我们还展示了 NHE1 依赖性 pHi 增加对细胞增殖、细胞周期进展和细胞迁移的功能意义。尽管 pHi 依赖性细胞功能具有广泛的意义，但我们对 pHi 变化如何影响蛋白质和大分子组装体的了解有限。为了解决这一限制，我们最近开始研究 pH 传感器的结构和功能，或者是具有对 pH 的微小生理变化敏感的活性或结合亲和力的蛋白质。当前的申请应用了我们在前四个资助周期中共同学到的知识，以确定调节癌细胞中异常的基本细胞过程的 pH 传感器的设计原理和功能。无论组织起源或遗传背景如何，pHi 升高是大多数癌症的标志。这可能反映出代谢适应、增殖和转移对较高 pHi 的依赖。我们将验证以下假设：控制新陈代谢和细胞周期进程的 pH 传感器在 NHE1 和 pHi 如何指导癌症中失调的细胞功能中发挥着关键作用。我们的研究采用创新的综合方法，将蛋白质结构、蛋白质生物化学和细胞生理学联系起来。在目标 1 中，我们将确定 NHE1 如何调节控制代谢的激酶。我们将根据我们最近的结构和生化发现（即其 p85 调节亚基是 pH 传感器）来测试对磷酸肌醇 3-激酶如何对 pH 敏感的预测。我们还将确定NHE1对磷酸果糖激酶1（PFK1）的调节，PFK1是糖酵解中的第一个限速酶，它直接结合NHE1的C端胞质结构域，并且具有极其pH敏感的活性。在目标 2 中，我们将重点关注我们之前的发现，即 NHE1 乘以 G2/M 时 H+ 流出，从而确定 NHE1 如何促进细胞增殖。我们将根据新的计算和生化数据以及缺乏 NHE1 活性的细胞中细胞周期蛋白 B1 表达如何减弱来测试对 Wee1 作为假定 pH 传感器的预测。在目标 3 中，我们将使用我们生成的果蝇模型确定 NHE 活性和 pHi 失调在肿瘤发生中的作用。我们将测试 Dnhe2 的缺失如何对转化细胞而非正常细胞造成合成致死的预测，并测试用目标 1 和 2 中生成的突变 pH 传感器对表型的拯救。我们还将询问 Dnhe2 的过度表达是否与癌基因激活或抑癌基因缺失协同作用以诱导转移性癌症，并将使用修饰筛选来识别 Dnhe2 过度表达的介质 表型。