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: 8389810
• 负责人: Anne M Collaco
• 金额: $ 5.82万
• 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/8389810
• 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采用组织和极化肠细胞来研究囊性纤维化跨膜电导调节因子（CFTR）阴离子通道的内化（内吞作用）及其胞吐插入顶端质膜（胞吐作用）如何调节其在肠中的表面表达和功能的小组。 这些研究对于肠生理学和囊性纤维化（CF）和分泌性腹泻的潜在病理生理学是至关重要的，因为：（1）肠表面上功能性CFTR的缺乏导致肠梗阻和CF，而（2）由于内吞作用的缺陷或胞吐作用的增加导致表面CFTR表达的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相反，内吞作用、胞吐作用和CFTR阴离子分泌由天然上皮细胞中的细胞类型特异性蛋白决定。网格蛋白介导CFTR内吞作用。我们实验室的研究确定了肌动蛋白运动肌球蛋白VI，作为CFTR内吞作用和肠道功能的关键调节因子。肌球蛋白VI不能直接结合CFTR。因此，我们检验了肠特异性衔接子将CFTR与网格蛋白连接的假设。我最初申请的中心目的是鉴定和表征负责CFTR的肌球蛋白VI依赖性内吞作用的肠特异性衔接子。由于对该申请进行了综述，我鉴定了负责CFTR内吞作用的第一个肠特异性衔接子（1-AP-2）（Collaco，A JBC; 285（22）pp 17177，2010）。因此，该修订的提议将（1）采用定点诱变、GST-下拉和酵母双杂交测定来确定1-AP-2与CFTR结合的位点（2）检查缺乏1-AP-2的极化肠细胞中CFTR表面表达、胞吞作用和阴离子分泌（3）检查1-AP-2在（i）募集CFTR和（ii）网格蛋白包被的小凹形成中的作用。计划进行为期三年的研究，以促进我在以下技能方面的培训：（a）Ussing室电生理学（B）酵母双杂交测定（3）慢病毒诱导极化上皮细胞中基因沉默（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，以提高手稿、赠款写作和演讲技巧。计划的课程，教学活动，每月与我的监督奖学金委员会的结构化会议，每周与我的导师和共同导师的会议以及与我的导师的日常互动将确保我在准备成为一名成功的独立调查员方面取得进展。