Role of LMTK2 in CFTR Trafficking

描述(申请人提供)：囊性纤维化(CF)是一种致命的遗传性疾病，仅在美国就有大约30,000人受到影响。囊性纤维化是由囊性纤维化跨膜电导调节基因突变引起的，该基因编码氯离子选择性阴离子通道。CFTR表达在呼吸道和胃肠道内极化上皮细胞的顶膜上，在那里它负责调节盐和水的运输。Cf中最常见的突变是由于Cftr中508(F508)位苯丙氨酸残基的缺失。？F508 CFTR在细胞表面的表达可以忽略不计，因为？F508 CFTR不能有效地从内质网输出，而且到达质膜的F508蛋白的半衰期严重减少。F508CFTR细胞表面稳定性的降低可能是由于内吞的F508CFTR不能适当地循环回质膜所致。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成纤维细胞等非生理性表达系统进行的。尽管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相关模型上皮细胞和组织的选择。然而，对极化上皮组织中CFTR的再循环知之甚少。缺乏这样的知识是一个重要的问题，因为如果没有这些知识，仅通过对生物合成途径的药理操作就不可能在细胞表面保持对治疗有益的水平？F508 CFTR。我们最近发现，LMTK2，一种与早期内小体相关的膜拴系激酶，对于引导内化的CFTR进入循环途径至关重要。我们目前的应用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)LMTK2活性的缺乏导致CFTR循环的丧失和细胞表面功能CFTR的丧失；(3)LMTK2的过表达导致CFTR循环的增强和细胞表面CFTR的增加。这项提议要检验的总体假设是，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的目标，不仅寻求表征LMTK2在调节CFTR贩运中的作用，而且寻求为我们的观察确定一个机制基础。我们相信，阐明CFTR贩运的机制将为以有益于治疗的方式加强？F508 CFTR循环提供新的治疗靶点。此外，我们确信，我们提出的研究不仅将为CFTR生物学提供重要的新见解，还将为蛋白质循环的机制提供重要的新见解。 公共卫生相关性：蛋白质运输异常是导致包括囊性纤维化(CF)在内的大量人类疾病的原因。了解CFTR(CF中的缺陷蛋白)运动中的分子事件将有助于确定治疗CF患者的新治疗靶点。

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.