ACTIONS OF THE SODIUM-H EXCHANGER SUBTYPE, NHE1

普遍表达的Na-H交换器NHE1的公认功能是通过细胞外Na+和细胞内H+的电子中和交换来调节细胞内的pH(Phi)。申请人的实验室最近确定，NHE1在调节肌动蛋白细胞骨架的组织中具有以前未被认识到的关键作用。整合素受体和GTP酶RhoA作用于NHE1的上游以刺激其活性。NHE1的激活反过来又是整合素和RhoA诱导的细胞骨架组织所必需的，包括肌动蛋白应激纤维的形成，局部黏附相关蛋白的表达、募集和组装，以及细胞在纤维连接蛋白基质上的附着和铺展速度。此外，NHE1的C末端胞质结构域被确定为直接与肌动蛋白结合蛋白ERM家族的成员结合，并且NHE1和ERM蛋白共定位于焦点接触部位。ERM蛋白，如NHE1，在RhoA反应的细胞骨架重塑中发挥关键作用，这些发现共同表明，NHE1可能通过提供将肌动蛋白拴在质膜上的结构链接来调节细胞骨架重塑。这一作用是否也涉及NHE1在离子转移中的既定作用仍未确定。这项建议的总体目标是通过研究NHE1提供作用于不同细胞骨架靶点的特定信号的假设，确定NHE1如何介导整合素和RhoA诱导的细胞骨架组织。目标1的研究将确定NHE1是否通过使用嵌合NHEs与ERM蛋白的结构关联来调节细胞骨架动力学，嵌合NHEs可以破坏或促进ERM结合。AIM 2中的研究将通过使用标记了pH敏感荧光探针的NHE1来显示局部的pH(I)梯度，以及通过确定易位缺陷的NHE1是否可以挽救NHE缺陷细胞中受损的细胞骨架重构，来确定离子转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细丝和控制细丝束的蛋白质的捆绑来研究NHE1的作用靶点。肌动蛋白细胞骨架的组织在一些正常的细胞过程中起着重要的作用，包括增殖、形态形成和伤口愈合，并与肿瘤转移和高血压等病理条件有关。确定NHE1是如何调控肌动蛋白细胞骨架的，将有助于我们理解离子交换器的功能重要性、肌动蛋白细丝组织的控制以及细胞骨架在细胞过程中所起的作用。

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.