Role of LMTK2 in CFTR Trafficking

LMTK2 在 CFTR 贩运中的作用

• 批准号: 8386586
• 负责人: Neil A Bradbury
• 金额: $ 36.65万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386586
• 关键词: Agonist; Anions; Back; Biochemical; Biological; Biology; Cell Line; Cell membrane; Cell surface; Cells; Chloride Ion; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Early Endosome; Endocytosis; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Epithelial; Epithelial Cells; Epithelium; Event; Fibroblasts; Gastrointestinal tract structure; Genotype; Half-Life; Hereditary Disease; Human; In Vitro; Individual; Intestines; Knowledge; Length; Lung diseases; Membrane; Modeling; Molecular; Monitor; Morbidity - disease rate; Movement; Mus; Mutate; Mutation; PC12 Cells; Pathway interactions; Patients; Phenylalanine; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Positioning Attribute; Proteins; Publications; Recycling; Regulation; Regulator Genes; Research; Role; Seminal; Signal Pathway; Site; Site-Directed Mutagenesis; Sodium Chloride; System; Testing; Tissues; United States; Water; apical membrane; base; cystic fibrosis patients; effective therapy; functional loss; human disease; in vivo; innovation; insight; mortality; mutant; new therapeutic target; novel; overexpression; protein transport; trafficking

Cystic fibrosis (CF) is a lethal genetic disease that afflicts some 30,000 individuals in the United States alone. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, that encodes for a chloride selective anion channel. CFTR is expressed in the apical membranes of polarized epithelial cells lining the airways and gastrointestinal tract, where it is responsible for regulating salt and water transport. The most common mutation in CF is caused by the deletion of a phenylalanine residue at position 508 (¿F508) in CFTR. Expression of ¿F508 CFTR at the cell surface is negligible because ¿F508 CFTR is not exported efficiently from the endoplasmic reticulum and because the half-life of ¿F508 protein that does reach the plasma membrane is severely reduced. The reduced cell surface stability of ¿F508 CFTR appears to be due to the inability of endocytosed ¿F508 CFTR to appropriately recycle back to the plasma membrane. The mechanisms and protein interactions that control the recycling of internalized CFTR are not known. Despite the many publications on CFTR trafficking, nearly all studies have been performed using non- physiological expression systems such as fibroblasts. Although these systems have proven easy to manipulate, their ease of use has been at the expense of physiological relevance. Critical to our present studies is the choice of relevant model epithelial cells and tissues. However, little is known about CFTR recycling in polarized epithelial tissues. Lack of such knowledge is an important problem, since without it, the ability to maintain therapeutically beneficial levels of ¿F508 CFTR at the cell surface solely through pharmacological manipulation of the biosynthetic pathway is unlikely. We have recently discovered that LMTK2, a membrane tethered kinase associated with early endosomes, is critical to directing internalized CFTR to the recycling pathway. The focus of our present application is based on three seminal observations; 1) CFTR is a substrate for LMTK2 kinase activity; (2) lack of LMTK2 activity leads to loss of CFTR recycling and a loss of functional CFTR from the cell surface; and (3) overexpression of LMTK2 leads to enhanced CFTR recycling and an increase in cell surface CFTR. The overall hypothesis to be tested in this proposal is that LMTK2 is a critical regulator in the endocytic recycling of CFTR. To test this hypothesis, we propose three specific aims that not only seek to characterize the role of LMTK2 in regulating CFTR trafficking, but also seek to identify a mechanistic basis for our observations. We are confident that elucidation of the mechanisms involved in CFTR trafficking will provide novel therapeutic targets for enhancing ¿F508 CFTR recycling in a therapeutically beneficial manner. In addition, we are certain that our proposed studies will provide important and novel insights not only into CFTR biology, but also into mechanisms of protein recycling.

囊性纤维化 (CF) 是一种致命的遗传性疾病，仅在美国就有约 30,000 人患病。 CF 是由囊性纤维化跨膜电导调节因子 (CFTR) 基因突变引起的， CFTR 编码氯选择性阴离子通道，在极化的顶膜中表达。 气道和胃肠道内壁的上皮细胞，负责调节盐和水 CF 中最常见的突变是由位置上苯丙氨酸残基的缺失引起的。 CFTR 中的 508 (¿F508) 的表达。细胞表面的 F508 CFTR 可以忽略不计，因为 ¿ F508 CFTR 是 不能有效地从内质网输出，并且因为 ¿ 的半衰期F508 蛋白的作用 到达质膜的能力严重降低，细胞表面稳定性降低。 F508 CFTR出现 是由于无法内吞 ¿ F508 CFTR 适当地回收回质膜。 控制内化 CFTR 循环的机制和蛋白质相互作用尚不清楚。 尽管有许多关于 CFTR 贩运的出版物，但几乎所有研究都是使用非 生理表达系统，例如成纤维细胞，尽管这些系统已被证明很容易实现。 操纵，它们的易用性以牺牲生理相关性为代价。 研究的重点是相关模型上皮细胞和组织的选择，然而，人们对CFTR知之甚少。 缺乏这种知识是一个重要的问题，因为如果没有它， 维持治疗有益水平的能力 ¿ F508 CFTR 在细胞表面仅通过 我们最近发现，不太可能对生物合成途径进行药理学操纵。 LMTK2 是一种与早期内体相关的膜栓激酶，对于指导内化至关重要 我们当前应用的重点基于三个开创性的观察： 1）CFTR是LMTK2激酶活性的底物；（2）LMTK2活性缺乏导致CFTR回收损失； (3) LMTK2 的过度表达导致细胞表面功能性 CFTR 的丧失； CFTR 回收和细胞表面 CFTR 的增加 本提案中要测试的总体假设是 LMTK2 是 CFTR 内吞再循环的关键调节因子。为了检验这一假设，我们提出。 三个具体目标不仅旨在描述 LMTK2 在监管 CFTR 贩运方面的作用，而且 我们有信心为我们的观察找到一个机制基础。 参与 CFTR 贩运将为增强 ¿ F508 CFTR 回收 此外，我们确信我们提出的研究将提供重要的帮助。 不仅对 CFTR 生物学，而且对蛋白质回收机制也有新的见解。