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：
8018340
负责人：
LOREN W RUNNELS
金额：
$ 10.18万
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：
2010
资助国家：
美国
起止时间：
2010-02-24 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8018340
关键词：
1-Phosphatidylinositol 3-Kinase Accounting Actomyosin Adhesions Adhesives Adult Affect Anterior Apical Biotinylation Blastopores Calcium Cell Adhesion Cell Line Cell membrane Cell physiology Cell surface Cells Data Defect Development Embryo Embryonic Development Event Extracellular Signal Regulated Kinases 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）：定向细胞运动对于具有适当极性（例如背腹、前后和左右对称）的生物体的发育是必需的。我们在非洲爪蟾中发现，TRPM7（第一个被发现具有自己的激酶结构域的离子通道）的耗尽会导致胚胎出现严重的原肠胚形成和神经折叠闭合缺陷，使 TRPM7 成为第一个被证明对早期脊椎动物具有显着影响的离子通道发展。对于这种效应的一个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-calpain 的活性来调节细胞粘附。尽管TRPM7在细胞运动中的作用已经有了令人信服的认识，但重要的细节仍然缺失，即TRPM7通道被激活的机制、激酶的调节，以及对TRPM7如何以及在什么条件下控制细胞运动的充分理解。最后，TRPM7 影响原肠胚形成的具体方面以及其激酶和通道在这些事件中发挥的作用尚未确定。我们提出了两个具体目标来阐明 TRPM7 在爪蟾发育过程中在细胞水平和体内的功能和调节。在第一个具体目标中，我们将采用电生理学方法来研究 TRPM7 通道的 PDGF 受体激活依赖于 PIP2 合成的假设。细胞表面生物素化实验将用于测试 PDGF 介导的 TRPM7 激活是否依赖于从细胞内位点向质膜招募通道。此外，我们还创建了 TRPM7 敲低的成纤维细胞系，以研究 TRPM7 激酶的调节及其磷酸化以及 PDGF 受体对肌球蛋白 II 的调节。最后，我们将测试PDGF受体是否利用TRPM7和ERK信号通路来调节m-钙蛋白酶和粘着斑周转。在第二个具体目标中，我们将采用我们以功能丧失/功能获得相结合的方法创建的通道和激酶死亡突变体来定义 TRPM7 通道和激酶在非洲爪蟾早期模式形成中的作用。这些研究将包括检查 TRPM7 对会聚伸展运动以及胚孔和神经折叠闭合的影响。总的来说，所提出的实验应该极大地促进我们对 TRPM7 体内功能的理解。对这种双功能通道的研究可以加深我们对包括神经嵴细胞迁移在内的许多生理过程的理解，并可能产生治疗依赖于细胞运动的病理状况的新策略，例如心脏病期间的炎症、癌细胞转移和脊髓损伤。