The CFTR-Associated Ligand (CAL) Arrests the Trafficking of the Mutant deltaF508 CFTR Channel in the ER Contributing to Cystic Fibrosis
MetadataShow full item record
Statement of Problem: ΔF508-CFTR is the most prevalent mutation among patients with Cystic Fibrosis. At present, there are no successful FDA-approved therapies for treatment. The CFTR-Associated Ligand (CAL) is a PDZ domain containing protein, which interacts with the C-terminus and CFTR. Here, we posit that CAL arrests trafficking of ΔF508-CFTR in the ER and as such regulates the cell surface expression of the mutant protein. Methods: Research for this dissertation was conducted using patient derived CFBE41o- cells that are stably transduced with either WT-CFTR or ΔF508-CFTR. CAL was overexpressed using an HA-tagged CAL plasmid and CAL was inhibited using a CAL specific siRNA. Experiments performed include western blotting, cell surface biotinylation, co-immunoprecipitation, confocal fluorescent microscopy, and the short circuit current assay. Results: CAL is localized to the endoplasmic reticulum (ER), is proteasomally degraded, and binds to ΔF508-CFTR in ΔF508-CFBE cells. Inhibition of CAL by siRNA caused the de novo production of a cell surface C band detected by cell surface biotinylation. This cell surface C band was shown to be functional using the short circuit current assay. Using microscopy, it was also shown that ΔF508-CFTR colocalizes with plasma membrane marker cadherin when CAL is inhibited. Additionally, CAL inhibition decreased binding of ΔF508-CFTR to VCP and increased ΔF508-CFTR binding to HSP70. Levels of NHERF1 increased in total lysate and ΔF508-CFTR colocalization with NHERF1 increased in ΔF508-CFBE cells. Overexpression of CAL caused the immature cell surface B band of ΔF508-CFTR to increase. This cell surface B band was shown to be nonfunctional. By microscopy, we noted that CAL overexpression enhances ΔF508-CFTR colocalization with ER marker, KDEL. Overexpression also increased the binding of ΔF508-CFTR to HSP40 and Aha1. NHERF1 levels decreased in total lysate, as did colocalization between NHERF1 and ΔF508-CFTR. Conclusions: CAL arrests trafficking of ΔF508-CFTR in the ER. When CAL is overexpressed, the sequestration of ΔF508-CFTR in the ER is enhanced but some nonfunctional ΔF508-CFTR escapes. When CAL is inhibited, ΔF508-CFTR is free to traffic through a conventional pathway to the cell surface where it successfully tranports chloride.