Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel found in epithelial cells. Previous studies from our laboratory have demonstrated that CFTR undergoes rapid endocytosis, suggesting that this provides a mechanism for regulating CFTR surface expression. In CF, more than 70% of patients have one specific mutation, deltaF508, which results in the production of a misfolded protein that fails to exit the endoplasmic reticulum (ER). An obvious therapeutic approach, therefore, is to develop methods for releasing deltaF508 CFTR to the cell surface, since deltaF508 has biological activity. Recent studies, however, have demonstrated the deltaF508 CFTR that reaches the cell surface after chemical chaperone or low temperature treatment is rapidly removed from the surface and degraded, unlike the wild type protein. Since little is known about how wild type CFTR surface expression is regulated, the defect in deltaF508 CFTR surface stability is unclear. Our hypothesis is that wild type CFTR is stabilized at the cell surface through interactions with epithelial-specific proteins such as EBP50, is internalized through clathrincoated pits because of sorting signals in its cytoplasmic tail regions, and is efficiently recycled back to the cell surface via vesicle trafficking. Further, we hypothesize that the phosphorylation status of CFTR regulates these processes and more importantly, mutations within CFTR dramatically influence CFTR trafficking and stability at the cell surface. To test these hypotheses, we will (1) define wild type CFTR trafficking in airway epithelial cells and (2) define how mutations in CFTR affect endocytosis and recycling. We will focus on three key aspects of CFTR cell biology: (1) plasma membrane residence time; (2) cellular mechanisms of endocytosis; and (3) degree of CFTR recycling. Using simplified biotinylation internalization assays of CFTR in human airway epithelial cells, we will assess the cell biological and physiological properties of CFTR trafficking of wild type CFTR versus a select panel of CFTR mutants that include deltaF508, R31L, Y1424A/I1427A, and deltaTRL. Understanding the normal dynamics of CFTR surface expression, internalization, and stability in epithelial cells where CFTR normally resides will provide valuable information regarding the fundamental cell biology of CFTR and will provide potential therapies for the most common mutation in CF.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK060065-04
Application #
6985397
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Mckeon, Catherine T
Project Start
2002-12-20
Project End
2007-11-30
Budget Start
2005-12-01
Budget End
2006-11-30
Support Year
4
Fiscal Year
2006
Total Cost
$283,185
Indirect Cost
Name
University of Alabama Birmingham
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Janaszak-Jasiecka, Anna; Bartoszewska, Sylwia; Kochan, Kinga et al. (2016) miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells. Sci Rep 6:22775
Fu, Lianwu; Rab, Andras; Tang, Li ping et al. (2015) ?F508 CFTR surface stability is regulated by DAB2 and CHIP-mediated ubiquitination in post-endocytic compartments. PLoS One 10:e0123131
Londino, James David; Lazrak, Ahmed; Noah, James W et al. (2015) Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection. FASEB J 29:2712-25
Bartoszewska, Sylwia; Kochan, Kinga; Piotrowski, Arkadiusz et al. (2015) The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1? expression in human endothelial cells through a negative feedback loop. FASEB J 29:1467-79
Matalon, Sadis; Bartoszewski, Rafal; Collawn, James F (2015) Role of epithelial sodium channels in the regulation of lung fluid homeostasis. Am J Physiol Lung Cell Mol Physiol 309:L1229-38
Collawn, James F; Matalon, Sadis (2014) CFTR and lung homeostasis. Am J Physiol Lung Cell Mol Physiol 307:L917-23
Madanecki, Piotr; Nozell, Susan; Ochocka, Renata et al. (2014) RNAdigest: a web-based tool for the analysis and prediction of structure-specific RNAse digestion results. PLoS One 9:e96759
Collawn, James F; Fu, Lianwu; Bartoszewski, Rafal et al. (2014) Rescuing ?F508 CFTR with trimethylangelicin, a dual-acting corrector and potentiator. Am J Physiol Lung Cell Mol Physiol 307:L431-4
Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta et al. (2013) Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity. Am J Physiol Lung Cell Mol Physiol 304:L582-92
Bartoszewska, Sylwia; Kochan, Kinga; Madanecki, Piotr et al. (2013) Regulation of the unfolded protein response by microRNAs. Cell Mol Biol Lett 18:555-78

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