Differential Localization of the Cystic Fibrosis Transmembrane Conductance Regulator in Normal and Cystic Fibrosis Airway Epithelium

Abstract

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…The ciliated cell is believed to be involved in transepithelial ion transport. Some reports suggest that CFTR is expressed in ciliated cells within the surface epithelium (Puchelle et al, 1992), consistent with a role for CFTR in regulating ASL volume. However, others report CFTR is expressed in airway surface goblet cells (Barasch et al, 1991;Jacquot et al, 1993;Glick et al, 2001), suggesting a regulatory role for CFTR in mucin secretion rather than fluid transport.…”

“…(Engelhardt et al, 1992(Engelhardt et al, , 1194Puchelle et al, 1992;Kalin et al, 1999;Penque et al, 2000). With the availability of very sensitive anti-CFTR mAbs (Mall et al, 2004) and LCM technologies, we initiated a comprehensive study to explore two major issues controversial in CF research: 1) the cell type specific localization of CFTR in the normal lung; and 2) the localization of ⌬F508 CFTR in native airway epithelium.…”

“…The maturation and localization of ⌬F508 CFTR protein in native human tissues, however, have been difficult to assess because of the limited sensitivity and specificity of CFTR antibodies and disease-related tissue damage. Early immunofluorescence studies described localization of WT but not ⌬F508 CFTR to the apical PM of the sweat duct (Cohn et al, 1991;Kartner et al, 1992) and airway submucosal glands (Engelhardt et al, 1992;Puchelle et al, 1992). Functional studies also failed to detect a cAMP-dependent Cl Ϫ secretion in native CF airway and intestinal epithelia (Boucher et al, 1989;Veeze et al, 1991;Mall et al, 2004).…”

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].…”

“…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].…”