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

Yale University, New Haven CT

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Abstract

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.