Role of LMTK2 in CFTR Trafficking

LMTK2 在 CFTR 贩运中的作用

• 批准号: 8039716
• 负责人: Neil A Bradbury
• 金额: $ 36.88万
• 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/8039716
• 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

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Protein trafficking abnormalities are responsible for a large number of human diseases including cystic fibrosis (CF). Understanding the molecular events in the movement of CFTR, the defective protein in CF, will allow the identification of novel therapeutic targets for the treatment of patients with CF.

描述（由申请人提供）：囊性纤维化（CF）是一种致命的遗传疾病，仅在美国就有大约30,000人患有此病。CF是由编码氯离子选择性阴离子通道的囊性纤维化跨膜传导调节（CFTR）基因突变引起的。CFTR在气道和胃肠道的极化上皮细胞的顶膜中表达，在那里它负责调节盐和水的运输。CF中最常见的突变是由508位苯丙氨酸残基的缺失引起的(？CFTR中F508)。的表达式？电池表面的F508 CFTR可以忽略不计，因为？F508 CFTR不能有效地从内质网输出，因为？到达质膜的F508蛋白严重减少。的细胞表面稳定性降低？F508 CFTR似乎是由于不能内吞？F508 CFTR适当回收回质膜。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，但几乎所有的研究都是使用非生理表达系统（如成纤维细胞）进行的。虽然这些系统已被证明易于操作，但它们的易用性是以牺牲生理相关性为代价的。我们目前研究的关键是选择相关的模型上皮细胞和组织。然而，我们对CFTR在极化上皮组织中的再循环知之甚少。缺乏这方面的知识是一个重要的问题，因为没有它，维持有益治疗水平的能力？F508 CFTR在细胞表面仅通过药理操作的生物合成途径是不可能的。我们最近发现LMTK2，一种与早期核内体相关的膜系链激酶，对于将内化的CFTR引导到循环途径至关重要。我们当前应用的重点是基于三个开创性的观察；1) CFTR是LMTK2激酶活性的底物；(2)缺乏LMTK2活性导致CFTR循环的丧失和细胞表面功能性CFTR的丧失；(3)过表达LMTK2导致CFTR循环增强，细胞表面CFTR增加。本提案要验证的总体假设是LMTK2是CFTR内吞循环的关键调节因子。为了验证这一假设，我们提出了三个具体目标，不仅试图表征LMTK2在调节CFTR贩运中的作用，而且还试图确定我们观察结果的机制基础。我们相信，对CFTR贩运机制的阐明将提供新的治疗靶点。F508 CFTR以治疗有益的方式回收。此外，我们相信我们提出的研究将不仅为CFTR生物学，而且为蛋白质循环机制提供重要和新颖的见解。