Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): Slow degradation of wild-type and ΔF508 CFTR in surface membrane preparations of immortalized airway epithelial cells

Abstract

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S] Met with chase periods of 5-24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 microM castanospermine, which is an inhibitor of processing alpha-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR.

High-Efficiency Transfer of Cystic Fibrosis Transmembrane Conductance Regulator cDNA into Cystic Fibrosis Airway Cells in Culture Using Lactosylated Polylysine as a Vector

“…A 50-fold increase in dark blue-staining cells, which represent a high expression of CFTR mRNA, was detected after transfection (Color Plate 2A) when compared with the cells that were treated with plasmid without lactosylated polylysine (Color Plate 2C). The number of moderately blue strained cells observed may represent endogenous CFTR, known to be present in these cells (Wei et al, 1996). The exon 14 sense probe showed only light blue stained cells (Color Plate 2B).…”

“…In situ detection of the CFTR protein was with antibody pAb3145 followed by development with horseradish peroxidase (HRP)-labeled anti-rabbit antibody, and diaminobenzidine (DAB) reagent as described (Mulberg et al, 1995;Wei et al, 1996). Transfection with plasmid alone served as a control.…”

Surfactant Protein A2 (SP-A2) Variants Expressed in CHO Cells Stimulate Phagocytosis of Pseudomonas aeruginosa More than Do SP-A1 Variants

“…Both CHO cell-and insect cell-expressed CFTR exhibited functional CFTR channel activity, but the activity of the insect-expressed CFTR was lower (1,33,48). The state of glycosylation of CFTR appeared to affect its stability at the plasma membrane (42,66).…”

Chloride conductance and genetic background modulate the cystic fibrosis phenotype of ΔF508 homozygous twins and siblings

“…However, the expression of ∆F508 CFTR in vivo might be different. Immunohistochemical studies performed on airway, intestinal, and hepatobiliary tissues (17,18,41,42) have demonstrated ∆F508 CFTR localization in the plasma membrane. Moreover, functional assays of transfected cells (43), in mice (44,45), and of human tissue specimens (18) have shown that ∆F508 CFTR is capable of transporting Clin response to cAMP agonists.…”