RUI: Regulation of MAP Kinase and NHE1 Activation by the G Proteins Gq and G13

钠氢交换异构体1(NHE1)存在于几乎所有哺乳动物细胞中，参与调节细胞内pH和细胞体积。NHE1活性可受多种细胞内信号通路的调节。激活通路的受体和参与调节的特定蛋白激酶在不同的细胞类型中差异很大。到目前为止，中国仓鼠肺成纤维细胞(CCL39)中的α1肾上腺素能信号通路尚无完整的研究。本项目的总体目标是阐明CCL39细胞中NHE1调控的信号通路。也是为了阐明哪些中间体参与了信号通路。具体目标1：研究PE对MAPK和NHE1的激活作用。初步研究表明，苯肾上腺素(PE)可同时激活MAPK和NHE1。研究MAPK激活的时间过程和剂量效应，以及MAPK激活与NHE1刺激之间的关系。将使用MAPK途径的特定激活剂和抑制剂来完成这一任务。这一目的将阐明MAPK作为PE刺激NHE的潜在关键中间体的作用。目的2：研究PKC和RAS在PE诱导的MAPK和NHE1活化中的作用。初步研究表明，PE对MAPK和PLD均有刺激作用。已发表的数据表明，1-肾上腺素能受体激活GQ。这种激活可以导致Q刺激PLCβ，并导致PKC的激活。此外，GQ。亚基可以导致RAS的激活。为了弄清PE调节NHE1活性的调控途径，将测定PKC和RAS激活的时间进程和剂量反应。然后将确定MAPK和NHE1的激活是否依赖于其中一个或两个中间体。表达显性/阴性RAS和/或显性/阴性Raf-1的转基因细胞将用于确定PE功能是否通过RAS依赖或独立机制激活MAPK和NHE1。这些蛋白的表达将在不同水平上扰乱假定的信号通路，澄清这些通路的汇聚点。这一目标将确定参与PE激活MAPK和NHE1的信号分子，并阐明PE激活的信号通路之间的关系。具体目标3：扩大本科生参与有意义的研究。穆尔黑德州立大学(MSU)生物和化学系教职员工的主要目的是进行研究项目，让本科生参与有意义的研究经历。学生们已经接受了培训，以执行所有必要的程序，以进行信号通路的调查，并测量提案中概述的NHE活动。这一研究项目将允许8-10名学生在学年期间参与研究，并允许三名学生在补助金所涵盖的三年中的夏季月份全职工作。

The Na + -H + exchanger isoform 1 (NHE1) is present in virtually all mammalian cells where it is involved in the regulation of intracellular pH (pHi) and cellular volume. NHE1 activity can be modulated by a wide variety of intracellular signaling pathways. The receptors that activate the pathways and the specific protein kinases involved in the regulation vary dramatically in different cell types. To date there has been no complete dissection of the a 1 adrenergic signaling pathway in Chinese hamster lung (CCL39) fibroblasts. The overall goal of this project is to clarify the signaling pathway for the regulation of NHE1 in CCL39 cells. Also to clarify which intermediates are involved in the signaling pathway. The following aims are proposed: Specific Aim 1: To study the activation of MAPK and NHE1 by PE. Preliminary studies indicate that phenylephrine (PE) can activate both MAPK and NHE1. The time course and the dose response MAPK activation will be investigated.The relationship between the activation of MAPK and the stimulation of NHE1 will also be investigated. Specific activators and inhibitors of the MAPK pathway will be used to accomplish this task. This aim will clarify the role of MAPK as a potential key intermediate in the stimulation of NHE by PE. Specific Aim 2: To study the involvement of PKC and Ras in the PE induced activation of MAPK and NHE1. Preliminary studies indicate that PE stimulates both MAPK and PLD. Published data indicates that a 1-adrenergic receptors activate Gq. This activation can lead to a q stimulating PLCbeta and leads to the activation of PKC. Additionally, the Gq . subunit can lead to the activation of Ras. To discern the regulatory pathways by which PE modulates NHE1 activity, the time course and dose response for activation of PKC and Ras will be determined. It will then be determined if MAPK and NHE1 activation dependent on one or both of the intermediates. Transfected cells expressing the dominant/negative Ras and/or dominant/negative Raf-1 will be used to determine if activation of MAPK and NHE1 by PE functions occurs through a Ras-dependent or independent mechanism. Expression of these proteins will disrupt the postulated signaling pathways at different levels clarifying the points of convergence of the pathways. This aim will determine the signaling molecules involved in the PE activation of MAPK and NHE1 and clarify the relationship between the signaling pathways activated by PE. Specific Aim 3: To expand the involvement of undergraduates in meaningful research. The primary purpose for faculty members of the biology and chemistry departments at Moorhead State University (MSU) is to have ongoing research projects that involve undergraduates in a meaningful research experience. Students have been trained to perform all the procedures required to carryout the investigations of the signaling pathways and to measure NHE activity as outlined in the proposal. This research project will allow 8 - 10 students to be involved in research during the academic year and three students to work full-time during the summer months of the three years covered by the grant.