Mislocalization of ΔF508 CFTR in cystic fibrosis sweat gland

Abstract

Misprocessing and mislocalization of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been described for the major CF-causing mutation (delta F508) in heterologous expression systems in vitro. We have generated monoclonal antibodies (mAbs) to CFTR with the aim of localizing the protein and its CF variants in vivo. Of the tissues where CFTR was observed, only the sweat gland is readily available and does not undergo secondary changes due to CF disease pathology. Sweat ducts from CF patients homozygous for delta F508 did not show the typical apical membrane staining seen in control biopsies. This demonstrates that the biosynthetic arrest and intracellular retention of delta F508 CFTR initially observed in vitro does occur in vivo and emphasizes the need to focus efforts on understanding the mislocalization.

Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

“…Accumulating evidence suggests that a fraction of CFTR channels resides in the early endosomal compartment, but its functional significance remains elusive [5][6][7][8][9][10][11]. Early endosomes comprise at least two distinct, but interconnected, populations of tubulovesicular structures, the sorting and recycling endosomes [39].…”

“…The efficiency of biotin stripping (92.2p2.3 %, n l 5) was evaluated by measuring the relative amount of biotinylated CFTR both before and after the reduction of the disulphide bonds on cells kept at 4 mC. Immunoprecipitation of the biotinylated CFTR was achieved with M3A7 and L12B4 monoclonal anti-CFTR antibodies [7] according to published protocol [25]. Immuno-isolated CFTR was eluted from protein G beads with 1 % (w\v) SDS.…”

“…Biotin molecules remaining at the cell surface were cleaved with the membrane-impermeant reducing agent MESNA. Biotinylated CFTR was immunoprecipitated sequentially with anti-CFTR antibody (M3A7 and L12B4 [7]) and streptavidin-Sepharose, and visualized by fluorography. Approximately 15 % (14.4p2.2 %, n l 3) of the cellsurface-labelled CFTR became endocytosed during the first three minutes of internalization (Figure 4), which was followed by a gradual loss of intracellular CFTR.…”

“…A growing body of evidence has corroborated the notion that translocation of CFTR from an internal vesicular pool may contribute to the cAMPstimulated plasma-membrane chloride conductance in certain cells. First, in addition to its plasma membrane localization, wild-type CFTR was detected in organelles along the endocytotic pathway by immunocytochemical analysis at the light-and electron-microscopy levels in secretory epithelia [5][6][7][8][9]. Secondly, CFTR conferred PKA-stimulated anion conductance to endosomal membranes both in non-polarized Chinese hamster ovary (CHO) cells transfected with CFTR cDNA and in T-84 gutepithelial cells expressing CFTR endogenously [10,11].…”

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

“…The most prevalent disease-causing mutation is ⌬F508, a deletion in exon 10 that results in loss of function due to misfolding and mislocalization of the mutant protein. [11][12][13][14][15] Crucial steps have been made in the last decade to show that a gene therapy approach to alleviating CF may be achievable. It is known that CFTR function can be restored in CF cell lines by delivering full length wild-type CFTR cDNA.…”