ACTIONS OF THE SODIUM-H EXCHANGER SUBTYPE, NHE1

普遍表达的Na-H交换器NHE 1的公认功能是通过细胞外Na+和细胞内H+的电中性交换来调节细胞内pH（pHi）。申请人的实验室最近确定，NHE 1在调节肌动蛋白细胞骨架的组织中具有先前未被认识到的关键作用。整合素受体和GTdR RhoA作用于NHE 1的上游以刺激其活性。NHE 1的激活反过来又是整联蛋白和RhoA诱导的细胞骨架组织所必需的，包括肌动蛋白应力纤维的形成，粘着斑相关蛋白的表达、募集和组装，以及细胞在纤连蛋白基质上附着和铺展的速率。此外，确定NHE 1的C-末端胞质结构域直接结合肌动蛋白结合蛋白的ERM家族的成员，并且NHE 1和ERM蛋白共定位于焦点接触位点。ERM蛋白，如NHE 1，在响应RhoA的细胞骨架重塑中起着关键作用，这些研究结果表明，NHE 1可能通过提供将肌动蛋白与质膜连接的结构连接来调节细胞骨架重塑。这一作用是否也涉及NHE 1在离子转运中的既定作用仍不确定。本提案的总体目标是确定NHE 1如何介导整合素和RhoA诱导的细胞骨架组织，通过调查NHE 1有助于作用于不同细胞骨架靶点的特定信号的假设。目标1中的研究将确定NHE 1是否通过使用破坏或促进ERM结合的嵌合NHE与ERM蛋白的结构关联来调节细胞骨架动力学。目标2中的研究将通过使用pH敏感性荧光探针标记的NHE 1来可视化局部pH（i）梯度，并通过确定易位缺陷的NHE 1是否可以挽救NHE缺陷细胞中受损的细胞骨架重塑，来确定离子易位在细胞骨架组织中的作用。目标3中的研究将通过关注肌动蛋白丝的成束和控制丝成束的蛋白质来研究NHE 1作用的靶点。肌动蛋白细胞骨架的组织化在调节许多正常细胞过程中起重要作用，包括增殖、形态发生和伤口愈合，并且它有助于病理状况，如肿瘤转移和高血压。确定NHE 1如何调节肌动蛋白细胞骨架将有助于我们理解离子交换剂的功能重要性，肌动蛋白丝组织的控制，以及细胞骨架在细胞过程中发挥的作用。

The recognized function of the ubiquitously expressed Na-H exchanger, NHE1, is to regulate intracellular pH (pHi) through electroneutral exchange of extracellular Na+ and intracellular H+. The applicant's laboratory recently determined that NHE1 has a previously unrecognized, critical role in regulating the organization of the actin cytoskeleton. Integrin receptors and the GTPase RhoA act upstream of NHE1 to stimulate its activity. Activation of NHE1, in turn is obligatory for integrin- and RhoA- induced cytoskeletal organization, including the formation of actin stress fibers, the expression, recruitment and assembly of focal adhesion-associated proteins, and the rate of cell attachment and spreading on a fibronectin matrix. Additionally, it was determined that the C-terminal cytoplasmic domain of NHE1 directly binds members of the ERM family of actin-binding proteins and that NHE1 and ERM proteins co-localize at sites of focal contact. ERM proteins, like NHE1, play a critical role in cytoskeletal remodeling in response to RhoA, and together these findings suggest that NHE1 may regulate cytoskeletal remodeling by providing a structural link to tether actin to the plasma membrane. Whether this action also involves the established role of NHE1 in ion translocation remains undetermined. The overall objective of this proposal is to determine how NHE1 mediates integrin- and RhoA- induced cytoskeletal organization by investigating the hypothesis that NHE1 contributes a specific signal that acts on distinct cytoskeletal targets. Studies in Aim 1 will determine whether NHE1 regulates cytoskeletal dynamics through a structural association with ERM proteins by using chimeric NHEs that either disrupt or promote ERM binding. Studies in Aim 2 will determine the role of ion translocation in cytoskeletal organization by using NHE1 tagged with pH-sensitive fluorescent probes to visualize localized pH(i) gradients, and by determining whether a translocation-defective NHE1 can rescue impaired cytoskeletal remodeling in NHE-deficient cells. Studies in Aim 3 will investigate the target of NHE1 actions by focusing on the bundling of actin filaments and proteins that control filament bundling. The organization of the actin cytoskeleton plays an important role in regulating a number of normal cellular processes, including proliferation, morphogenesis and wound healing, and it contributes to pathological conditions such as tumor metastasis and hypertension. Determining how NHE1 regulates the actin cytoskeleton will contribute to our understanding of the functional importance of ion exchangers, the control of actin filament organization, and the role the cytoskeleton plays in cellular processes.