Mechanisms of Receptor Regulated Na+-H+ Exchange

描述(申请人提供)：普遍表达的Na-H交换器NHE1通过催化细胞外Na+与细胞内H+的电中和交换，在细胞内pH和细胞体积动态平衡方面具有既定的功能。在过去的资助期间，确定了NHEI作为肌动蛋白细丝锚的辅助功能。NHE1的肌动蛋白锚定是由ERM(Ezrin，Radioxin，Moesin)肌动蛋白结合蛋白直接结合介导的，在成纤维细胞中，NHEI是保持NHEI在板脂远端边缘的优势定位，正常的细胞形状，以及皮质肌动蛋白细丝的组装所必需的。目前建议的目标是建立NHEI的额外辅助功能，作为组装信号综合体和促进信号传递的脚手架平台。NHE1的C末端胞质结构域直接与至少10个不同功能的信号分子结合，这些分子协调调节NHE1的活性，限制NHE1在专门的膜域中的定位，并如现在所提出的那样，可能向不同的效应器通路传递信号。着眼于NHE1在成纤维细胞中的作用，该方案研究了NHE1作为空间受限的支架来组装信号复合体和促进信号传递的假说。目标1的研究将通过询问NHE1的支架如何决定其定位，以及NHE1在片状脂体中的定位如何决定其与已鉴定的相互作用蛋白的结合，来确定NHE1的支架和定位是如何整合的。目标2的研究将测试NHE1支架在信号复合体组装和信号传递中的作用。不同的信号单元，每个都有一个直接与NHE1结合的组件，将通过询问是否与NHE1结合，NHE1在片状脂膜中的定位，或者NHE1的离子转移对于单元的组装和信号传递是必要的来进行研究。具有代表性的信号单位包括Ste20-1ike激酶Nik对Ezrin和Arp2/3的磷酸化，Ezrin与GTP酶Rac和CDc42的结合，以及Rho激酶ROCK对肌球蛋白轻链的磷酸化。目标3中的研究将通过使用蛋白质组学方法来确定NHE1支架整体的动态组成，从而定义NHE1支架的复杂性，然后询问当NHE1定位或离子移位受损时，相关蛋白质的图谱是否不同。NHEI在细胞内pH动态平衡、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。

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.