Elucidating the role of a-AP-2 in CFTR endocytosis and function in the intestine

描述(申请人提供)：我们实验室是唯一一个利用组织和极化的肠道细胞来研究囊性纤维化跨膜电导调节器(CFTR)阴离子通道的内化(内吞作用)及其胞外插入到顶端质膜(胞吐作用)如何调节其在肠道中的表面表达和功能的团队。这些研究对于囊性纤维化(CF)和分泌性腹泻的肠道生理学和基本病理生理学是至关重要的，因为：(1)肠道表面缺乏功能性CFTR会导致肠梗阻和CF，而(2)由于内吞作用缺陷或胞吐作用增加而导致表面CFTR表达增加，从而导致分泌性腹泻。与在人工过度表达和非极化细胞模型中观察到的相反，在天然上皮细胞中，内吞、胞吐和CFTR阴离子分泌是由细胞类型特定的蛋白质决定的。笼状蛋白介导CFTR内吞作用。我们实验室的研究证实，肌动蛋白肌球蛋白VI是CFTR内吞作用和肠道功能的关键调节因子。肌球蛋白VI不能直接与CFTR结合。因此，我们检验了一种假设，即肠道特异的接头会将cftr与笼状蛋白联系起来。我最初应用的中心目标是鉴定和表征负责CFTR依赖肌球蛋白VI的内吞作用的肠道特异性接头。自从该应用程序被审查以来，我确定了第一个负责CFTR内吞的肠道特异性适配器(1-AP-2)(Collaco，A JBC；285(22)pp17177,2010)。因此，这项修订建议将(1)使用定点突变、GST-下拉和酵母两种杂交试验来确定1-AP-2与CFTR结合的位置(2)检测缺乏1-AP-2的极化肠细胞的CFTR表面表达、内吞作用和阴离子分泌(3)检测1-AP-2在(I)招募CFTR和(Ii)网状蛋白包裹的凹陷形成中的作用。拟议的研究计划在三年内进行，以促进我在以下技能方面的培训：(A)使用小室电生理学(B)酵母双杂交分析(3)Lenti病毒诱导极化上皮细胞基因沉默(3)冷冻免疫电子显微镜和(4)透射电子显微镜。如果我想成为一名成功的独立调查员，我还需要进一步的培训来提高手稿、赠款写作和陈述技能。计划的课程、教学活动、与我的监督奖学金委员会的有组织的月度会议、与我的导师和合作导师的每周会议以及与我的导师的日常互动，都将确保在为我成为一名成功的独立调查员做准备方面取得进展。

DESCRIPTION (provided by applicant): Our laboratory is the only group that employs tissues and polarized intestinal cells to examine how internalization (endocytosis) of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channels and its exocytic insertion back to the apical plasma membrane (exocytosis) regulate its surface expression and function in the intestine. These investigations are critical to intestinal physiology and the underlying pathophysiology of Cystic Fibrosis (CF) and secretory diarrhea because: (1) absence of functional CFTR on the intestinal surface leads to intestinal obstruction and CF while (2) increased surface CFTR expression due to defects in endocytosis or increased exocytosis result in secretory diarrhea. In contrast to what is observed in artificial over-expression and non-polarized cell models, endocytosis, exocytosis and CFTR anion secretion are dictated by cell-type specific proteins in native epithelial cells. Clathrin mediates CFTR endocytosis. Studies from our laboratory identified the actin motor myosin VI, as a key regulator of CFTR endocytosis and function in the intestine. Myosin VI is unable to directly bind CFTR. Thus, we examined the hypothesis that an intestine specific adaptor would link CFTR to clathrin. The central aim of my initial application was to identify and characterize the intestine specific adaptor responsible for myosin VI- dependent endocytosis of CFTR. Since the application was reviewed, I identified the first intestine- specific adaptor (1-AP-2) responsible for CFTR endocytosis (Collaco, A JBC; 285 (22) pp17177, 2010). Accordingly, this revised proposal will (1) employ site-directed mutagenesis, GST-pull down and yeast two hybrid assays to determine the sites of 1-AP-2 binding to CFTR (2) examine CFTR surface expression, endocytosis and anion secretion in polarized intestinal cells lacking 1-AP-2 (3) examine role of 1-AP-2 in (i) recruiting CFTR and (ii) clathrin-coated pit formation. The proposed studies are planned over a three- year period in order to facilitate my training in the following skills: (a) Ussing chamber electrophysiology (b) yeast two-hybrid assays (3) lenti-virus induced silencing of genes in polarized epithelial cells (3) cryo- immunoelectron microscopy and (4) transmission electron microscopy. I also need further training to enhance manuscript, grant writing and presentation skills if I am to emerge as a successful independent investigator. The courses planned, teaching activities, structured monthly meetings with my oversight scholarship committee, weekly meetings with my mentor and co-mentor and daily interactions with my mentor will ensure that progress is achieved in preparing me to emerge as a successful independent investigator.