Role of LMTK2 in CFTR Trafficking

囊性纤维化(CF)是一种致命的遗传病，仅在美国就有约30,000人受到影响。 囊性纤维化是由囊性纤维化跨膜传导调节基因突变引起的， 编码用于氯离子选择性阴离子通道。Cftr在极化的根尖膜中表达 位于呼吸道和胃肠道的上皮细胞负责调节盐分和水分。 运输。Cf中最常见的突变是由位置上的苯丙氨酸残基缺失引起的 508(？F508)。F508 CFTR在细胞表面的表达可以忽略不计，因为F508 CFTR是 不能有效地从内质网输出，这是因为F508蛋白的半衰期 到达质膜是严重减少的。F508 CFTR细胞表面稳定性降低 这是由于内吞的F508 CFTR不能适当地循环回到质膜。 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。尽管事实证明，这些系统很容易 操纵，它们的易用性一直是以牺牲生理相关性为代价的。对我们现在来说至关重要 研究内容是选择相关的模型上皮细胞和组织。然而，人们对CFTR知之甚少 极化上皮组织中的循环。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， 仅通过以下途径在细胞表面保持对治疗有益的F508 CFTR水平的能力 对生物合成途径进行药理学操作是不可能的。我们最近发现， LMTK2是一种与早期内小体相关的膜拴系激酶，对内化内化的导向至关重要。 Cftr到回收路径。我们目前申请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)LMTK2活性的缺乏导致CFTR循环的丧失 以及(3)过表达LMTK2导致细胞功能增强 CFTR回收和细胞表面CFTR的增加。这项提案中要检验的总体假设是 提示LMTK2是CFTR胞内循环的关键调控因子。为了检验这一假设，我们建议 三个具体目标，不仅试图确定LMTK2在管制CFTR贩运方面的作用，而且还 试图为我们的观察找出一个机械的基础。我们相信，对这些机制的解释 参与CFTR贩运将提供新的治疗目标，以加强F508 CFTR在 有益于治疗的方式。此外，我们确信，我们提议的研究将提供重要的 不仅对CFTR生物学，而且对蛋白质循环的机制也有新的见解。

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.