Mechanisms of Receptor Regulated Na+-H+ Exchange

描述（由申请人提供）：广泛表达的Na-H交换剂NHE 1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既定功能。在过去的资助期间，NHEI作为肌动蛋白丝的锚的辅助功能被确定。肌动蛋白锚定由NHE 1介导的直接结合的ERM（埃兹蛋白，radixin，膜突蛋白）肌动蛋白结合蛋白，并在成纤维细胞是必要的，以保持主要的本地化NHEI在板状伪足，正常的细胞形状，和皮质肌动蛋白丝的装配的远端边缘。目前建议的目的是为国家高等教育机构建立一个额外的辅助功能，作为组装信号综合体和促进信号传递的脚手架平台。NHE 1的C-末端胞质结构域直接结合至少10种功能不同的信号分子，这些信号分子协调调节NHE 1活性，限制NHE 1在专门的膜结构域中的定位，并且如现在提出的，可以将信号传递到不同的效应子途径。聚焦于NHE 1在成纤维细胞中的作用，该提议调查了NHE 1作为空间限制的支架组装信号复合物并促进信号中继的假设。目标1中的研究将通过询问NHE 1的支架如何决定其定位以及NHE 1在板状伪足中的定位如何决定其与鉴定的相互作用蛋白的结合来确定NHE 1支架和定位是如何整合的。目标2中的研究将测试NHE 1支架在信号复合物组装和信号中继中的作用。不同的信号单元，每个都有一个组件，直接结合到NHE 1，将通过询问是否结合到NHE 1，NHE 1在板状伪足的本地化，或离子易位NHE 1是必要的组装和信号中继的单位。代表性的信号传导单元包括通过Ste 20样激酶NIK磷酸化ezrin和Arp 2/3，ezrin与GTP酶Rac和Cdc 42结合，以及通过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NHE 1支架集合的动态组成来定义NHE 1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的谱是否不同。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理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.