Cystic fibrosis (CF) is a lethal genetic disease that afflicts some 30,000 individuals in the United States alone. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes for a chloride selective anion channel. CFTR is expressed in the apical membranes of polarized epithelial cells lining the airways and gastrointestinal tract, where it is responsible for regulating salt and water transport. The most common mutation in CF is caused by the deletion of a phenylalanine residue at position 508 (?F508) in CFTR. Expression of ?F508 CFTR at the cell surface is negligible because ?F508 CFTR is not exported efficiently from the endoplasmic reticulum and because the half-life of ?F508 protein that does reach the plasma membrane is severely reduced. The reduced cell surface stability of ?F508 CFTR appears to be due to the inability of endocytosed ?F508 CFTR to appropriately recycle back to the plasma membrane. The mechanisms and protein interactions that control the recycling of internalized CFTR are not known. Despite the many publications on CFTR trafficking, nearly all studies have been performed using non-physiological expression systems such as fibroblasts. Although these systems have proven easy to manipulate, their ease of use has been at the expense of physiological relevance. Critical to our present studies is the choice of relevant model epithelial cells and tissues. However, little is known about CFTR recycling in polarized epithelial tissues. Lack of such knowledge is an important problem, since without it, the ability to maintain therapeutically beneficial levels of ?F508 CFTR at the cell surface solely through pharmacological manipulation of the biosynthetic pathway is unlikely. We have recently discovered that LMTK2, a membrane tethered kinase associated with early endosomes, is critical to directing internalized CFTR to the recycling pathway. The focus of our present application is based on three seminal observations;1) CFTR is a substrate for LMTK2 kinase activity;(2) lack of LMTK2 activity leads to loss of CFTR recycling and a loss of functional CFTR from the cell surface;and (3) overexpression of LMTK2 leads to enhanced CFTR recycling and an increase in cell surface CFTR. The overall hypothesis to be tested in this proposal is that LMTK2 is a critical regulator in the endocytic recycling of CFTR. To test this hypothesis, we propose three specific aims that not only seek to characterize the role of LMTK2 in regulating CFTR trafficking, but also seek to identify a mechanistic basis for our observations. We are confident that elucidation of the mechanisms involved in CFTR trafficking will provide novel therapeutic targets for enhancing ?F508 CFTR recycling in a therapeutically beneficial manner. In addition, we are certain that our proposed studies will provide important and novel insights not only into CFTR biology, but also into mechanisms of protein recycling.
Protein trafficking abnormalities are responsible for a large number of human diseases including cystic fibrosis (CF). Understanding the molecular events in the movement of CFTR, the defective protein in CF, will allow the identification of novel therapeutic targets for the treatment of patients with CF.
|Bradbury, Neil (2017) Do Students Really Have an Inability to Concentrate During Lectures? Acad Med 92:428|
|Dey, Isha; Shah, Kalpit; Bradbury, Neil A (2016) Natural Compounds as Therapeutic Agents in the Treatment Cystic Fibrosis. J Genet Syndr Gene Ther 7:|
|Shah, Kalpit; Bradbury, Neil A (2015) Kinase modulation of androgen receptor signaling: implications for prostate cancer. Cancer Cell Microenviron 2:|
|Jai, Ying; Shah, Kalpit; Bridges, Robert J et al. (2015) Evidence against resveratrol as a viable therapy for the rescue of defective ?F508 CFTR. Biochim Biophys Acta 1850:2377-84|
|Butler, E C; Bradbury, Neil A (2015) Signal dependent ER export of lemur tyrosine kinase 2. BMC Cell Biol 16:26|
|Shah, Kalpit; Cheng, Yi; Hahn, Brian et al. (2015) Synonymous codon usage affects the expression of wild type and F508del CFTR. J Mol Biol 427:1464-1479|
|White, Carl; Nixon, Alex; Bradbury, Neil A (2015) Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP). J Vis Exp :|
|Shah, Kalpit; McCormack, Charles E; Bradbury, Neil A (2014) Do you know the sex of your cells? Am J Physiol Cell Physiol 306:C3-18|
|Nixon, Alexander; Jia, Ying; White, Carl et al. (2013) Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection. Am J Physiol Cell Physiol 304:C164-9|