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

阐明 a-AP-2 在 CFTR 内吞作用和肠道功能中的作用

• 批准号: 8494613
• 负责人: Anne M Collaco
• 金额: $ 5.99万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-08 至 2014-06-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8494613
• 关键词: Actins; Adaptor Signaling Protein; Anions; Apical; Back; Bicarbonates; Binding; Binding Proteins; Biological Assay; Biotinylation; Cell membrane; Cell model; Cell surface; Cells; Chimeric Proteins; Chloride Ion; Chlorides; Clathrin; Clathrin-Coated Vesicles; Communities; Cyclic Nucleotides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Diarrhea; Disease; Educational process of instructing; Electrophysiology (science); Endocytosis; Ensure; Enterocytes; Epithelial Cells; Excision; Exocytosis; Fluids and Secretions; Functional disorder; Gene Silencing; Grant; Health; Immunoelectron Microscopy; Integral Membrane Protein; Intestinal Diseases; Intestinal Obstruction; Intestines; Investigation; Ion Channel; Ion Transport; Laboratories; Link; Manuscripts; Mediating; Membrane; Mentors; Motor; Physiological; Physiology; Proteins; Publishing; Recruitment Activity; Research Personnel; Role; Scholarship; Site; Site-Directed Mutagenesis; Small Intestines; Structure; Subfamily lentivirinae; Surface; TFAP2A gene; Tissues; Training; Transmission Electron Microscopy; Two-Hybrid System Techniques; Up-Regulation; Writing; Yeasts; apical membrane; cell type; cellular microvillus; coated pit; density; meetings; myosin VI; polarized cell; receptor; skills; small hairpin RNA

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.

描述（由申请人提供）：我们的实验室是唯一一个利用组织和极化肠细胞来研究囊性纤维化跨膜传导调节剂（CFTR）阴离子通道的内化（内吞作用）及其胞外插入回顶端质膜（胞外作用）如何调节其在肠道中的表面表达和功能的团队。这些研究对于肠道生理学和囊性纤维化（CF）和分泌性腹泻的潜在病理生理学至关重要，因为：(1)肠道表面功能性CFTR缺失导致肠梗阻和CF，(2)由于内吞缺陷或胞吐增加导致表面CFTR表达增加导致分泌性腹泻。与在人工过表达和非极化细胞模型中观察到的情况相反，天然上皮细胞的内吞作用、胞吐作用和CFTR阴离子分泌是由细胞类型特异性蛋白决定的。网格蛋白介导CFTR内吞作用。我们实验室的研究发现肌动蛋白运动肌球蛋白VI是CFTR在肠道内吞作用和功能的关键调节因子。Myosin VI不能直接结合CFTR。因此，我们检验了肠道特异性接头将CFTR与网格蛋白连接起来的假设。我最初申请的中心目的是确定和表征负责肌球蛋白VI依赖性CFTR内吞作用的肠道特异性接头。自审查申请以来，我确定了第一个负责CFTR内吞作用的肠道特异性适配器（1-AP-2） （Collaco, A JBC; 285 (22) pp17177, 2010）。因此，本修订方案将(1)采用定点诱变、GST-pull - down和酵母两种杂交试验来确定1- ap -2与CFTR结合的位点(2)在缺乏1- ap -2的极化肠细胞中检测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）网格蛋白包被窝形成中的作用。拟议的研究计划为期三年，以促进我在以下技能方面的培训：(a)使用室电生理学(b)酵母双杂交试验(3)慢病毒诱导极化上皮细胞中基因沉默(3)冷冻免疫电镜和(4)透射电镜。如果我要成为一名成功的独立调查员，我还需要进一步的培训，以提高手稿、拨款写作和演讲技巧。课程规划、教学活动、与奖学金监督委员会的月度例会、与导师的每周例会以及与导师的日常互动，都将确保我在成为一名成功的独立研究者的过程中取得进展。