Investigation of CFTR intermolecular structure and protein-protein interactions

Cystic fibrosis (CF) is a common heritable disorder primarily affecting of people of northern European descent. It is caused by mutations in the C&barbelow;ystic F&barbelow;ibrosis T&barbelow;ransmembrane conductance R&barbelow;egulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. CFTR functions in the epithelial layer of cells of the intestine, pancreas and lung. Most commonly the disease is caused by defective intracellular trafficking of mutant CFTR channels, resulting in little or no functional channels being localized to the plasma membrane layer of cells. A single mutation ΔF508 accounts for over 70% of the cases of cystic fibrosis. The ΔF508 CFTR mutant protein is characterized by defective trafficking in which less than 1% of the mutant channels reach their proper destination in the plasma membrane. In addition, wildytpe CFTR channels are inefficiently trafficked, with much of the synthesized protein being degraded before it leaves the endoplasmic reticulum (ER).; The presence of trafficking signals necessary for efficient trafficking through the early protein secretory pathway has been demonstrated for several proteins. These protein trafficking signals usually consist of short amino acid motifs that interact with proteins important in the secretory pathway. We have investigated the amino-terminus of CFTR and identified several potential trafficking signals. A di-phenylalanine (FF) motif within the CFTR amino terminus is necessary for efficient CFTR trafficking to the Golgi and interacts with a protein component of COP I vesicles, that traffic in an antergrade and retrograde fashion within the ER and Golgi compartments. In addition, a vesicle protein component of COP II vesicles that bud from the ER, interacts with the CFTR amino terminus. COP II proteins have been demonstrated to interact with a di-acidic (Asp-X-Glu) motif that is also located within the CFTR amino-terminus.; The stoichiometry of the CFTR channel has been investigated, and although the minimal functional unit is the monomer, the possibility of a multimeric structure has not been ruled out. Using a co-expression and “pull-down” assay, we have demonstrated that CFTR does indeed form a multimer in mammalian cells. Further, we demonstrate that “wildtype” CFTR interacts with ΔF508 CFTR and that the interaction has the effect of promoting the normally defective ΔF508 protein to traffic to the plasma membrane. Our results are consistent with a dimeric structure for the CFTR channel.; We have previously demonstrated using a yeast-two hybrid system that we detect dimers between a cytosolic domain of CFTR, the nucleotide binding domain 1 (NBD1). When wildtype NBD1 is used we detect an interaction, but when the ΔF508 mutation is included no interaction is detected. We further characterize this interaction through the identification of sub-domains of NBD1 using truncations that prevent the yeast two-hybrid interaction. Finally, we identify new second site revertants of ΔF508 that restore dimerization when the ΔF508 mutation is included in the NBD1. We then characterize the new revertants in full length CFTR ΔF508 channels for their ability to restore CFTR channel processing and function in mammalian cells.