Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines

• Published in: The Journal of biological chemistry 2004-05, Vol.279 (21), p.22578-22584
• Main Authors: Varga, Károly, Jurkuvenaite, Asta, Wakefield, John, Hong, Jeong S, Guimbellot, Jennifer S, Venglarik, Charles J, Niraj, Ashutosh, Mazur, Marina, Sorscher, Eric J, Collawn, James F, Bebök, Zsuzsa
• Format: Article
• Language: English
• Subjects: Animals; Biotinylation; Cell Line; Cell Membrane - metabolism; COS Cells; Cysteine Endopeptidases; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Endoplasmic Reticulum - metabolism; Epithelial Cells - metabolism; Glycosylation; Golgi Apparatus - metabolism; HeLa Cells; Humans; Kinetics; Multienzyme Complexes - antagonists & inhibitors; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M401522200

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, ∼75% of wild type CFTR is degraded by the e ndoplasmic r eticulum (ER)- a ssociated d egradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are ∼4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.