Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

双功能离子通道和激酶 TRPM7 的功能分析

基本信息

批准号：
8047995
负责人：
LOREN W RUNNELS
金额：
$ 29.05万
依托单位：
UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8047995
关键词：
Accounting Actomyosin Adhesions Adhesives Adult Affect Anterior Apical Biotinylation Blastopores Calcium Calpain II Cell Adhesion Cell Line Cell membrane Cell physiology Cell surface Cells Data Defect Development Embryo Embryonic Development Event Fibroblasts Figs - dietary Focal Adhesions Heart Diseases Hydrolysis Inflammation Investigation Ion Channel Lead Left Lysophosphatidic Acid Receptors Lysophospholipids Mediating Mitogen-Activated Protein Kinases Modeling Movement Myosin Type II Nature Neoplasm Metastasis Neural Crest Cell Neural Fold Oligonucleotides Organism Paper Pathway interactions Pattern Formation Peptide Hydrolases Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphorylation Phosphotransferases Physiological Processes Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Play Property Proteins Publishing Receptor Activation Regulation Reporting Research Personnel Role Signal Pathway Signal Transduction Pathway Site Spinal cord injury Testing Time Tissues Work Xenopus laevis cancer cell cell motility constriction directional cell gain of function gastrulation in vivo inorganic phosphate loss of function lysophosphatidic acid m-calpain mutant research study xenopus development

项目摘要

DESCRIPTION (provided by applicant): Directional cell motility is required for the development of an organism with proper polarity such as dorso-ventral, anterior-posterior, and left-right symmetry. We have found in Xenopus laevis that depletion of TRPM7, the first ion channel discovered to have its own kinase domain, results in embryos with severe gastrulation and neural fold closure defects, making TRPM7 the first ion channel shown to have a dramatic effect on early vertebrate development. A possible explanation for this effect is our recently reported discovery that TRPM7 controls the activity of the calcium-dependent protease m-calpain to regulate cell adhesion. Although a compelling picture is emerging of TRPM7's role in cell motility, important details are still missing, namely, the mechanism by which TRPM7's channel is activated, regulation of the kinase, and a full understanding of how and under what conditions TRPM7 controls cell motility. Finally, the specific aspect(s) of gastrulation affected by TRPM7 and the roles played by its kinase and channel in these events have not been defined. We propose two specific aims to clarify TRPM7's function and regulation on the cellular level and in vivo during Xenopus development. In the first specific aim, we will take an electrophysiological approach to investigate the hypothesis that PDGF-receptor activation of TRPM7's channel is dependent upon PIP2 synthesis. Cell surface biotinylation experiments will be used to test whether PDGF-mediated activation of TRPM7 relies upon the recruitment of the channel to the plasma membrane from intracellular sites. In addition, we've created TRPM7-knockdown fibroblast cell lines to investigate the regulation of TRPM7's kinase and its phosphorylation and regulation of myosin II by the PDGF receptor. Finally, we will test whether the PDGF receptor utilizes both TRPM7 and the ERK signaling pathway to regulate m-calpain and focal adhesion turnover. In the second specific aim we will employ channel- and kinase-dead mutants we've created in a combined loss-of-function/gain-of-function approach to define the roles of TRPM7's channel and kinase in early pattern formation in Xenopus laevis. These investigations will include an examination of TRPM7's influence on convergent extension movements and blastopore and neural fold closure. Collectively, the proposed experiments should greatly advance our understanding of TRPM7's function in vivo. Study of this bifunctional channel could deepen our understanding of many physiological processes including neural crest cell migration and could potentially lead to new strategies for treating pathological conditions dependent on cell motility such as inflammation during heart disease, cancer cell metastasis, and spinal cord injuries.

描述（由申请人提供）：具有适当极性的生物（例如腹侧，前后和左右对称性）的生物的发展需要定向细胞运动。我们在Xenopus laevis中发现TRPM7的耗竭是第一个离子通道被发现具有自己的激酶结构域，导致具有严重胃肠道和神经折叠缺陷的胚胎，这使得TRPM7使TRPM7成为第一个离子通道，显示出对早期脊椎动物发育产生巨大效应。对此效果的一种可能的解释是我们最近报道的发现，TRPM7控制钙依赖性蛋白酶M-钙蛋白的活性以调节细胞粘附。尽管引人注目的图片是TRPM7在细胞运动中的作用的出现，但重要的细节仍然缺少，即，激活TRPM7通道的机制，激酶的调节以及对TRPM7在什么条件下以及在什么情况下控制细胞运动的充分理解。最后，尚未定义受TRPM7影响的胃结构的特定方面以及其激酶和渠道在这些事件中扮演的角色。我们提出了两个具体的目的，以阐明在爪蟾发育过程中TRPM7对细胞水平和体内的功能和调节。在第一个具体目的中，我们将采用一种电生理方法来研究TRPM7通道的PDGF受体激活的假设取决于PIP2的合成。细胞表面生物素化实验将用于测试PDGF介导的TRPM7的激活是否依赖于从细胞内部位募集到质膜的通道。此外，我们还创建了TRPM7-KNOCKDOWN成纤维细胞系，以研究PDGF受体对TRPM7激酶的调节及其对肌球蛋白II的磷酸化和调节。最后，我们将测试PDGF受体是否同时利用TRPM7和ERK信号通路来调节M-粘蛋白和局灶性粘附周转率。在第二个特定目的中，我们将采用我们在综合功能丧失/功能获得方法中创建的通道和激酶死亡突变体，以定义Xenopus laevis早期模式形成中TRPM7通道和激酶在早期模式形成中的作用。这些研究将包括对TRPM7对收敛延伸运动的影响以及胚胎和神经折叠的影响。总的来说，提出的实验应大大提高我们对TRPM7在体内功能的理解。对这种双功能通道的研究可能会加深我们对许多生理过程的理解，包括神经rest细胞迁移，并有可能导致新的策略来治疗依赖细胞运动的病理状况，例如心脏病期间的炎症，癌细胞转移，脊髓和脊髓损伤。