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

Abstract

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, ϳ75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (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 ϳ4-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.

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

“…epithelial cells in which endogenous CFTR is studied have less band B protein than that detected when recombinant CFTR is expressed (31,32). Treatment with the miR-138 mimic and the SIN3A DsiRNA increased cAMP-stimulated G t (Fig.…”

Protein synthesis inhibitors and the chemical chaperone TMAO reverse endoplasmic reticulum perturbation induced by overexpression of the iodide transporter pendrin

“…A recent study has shown that inefficient processing of CFTR occurs only in heterologous overexpression systems. In epithelial cells, endogenously expressed CFTR is efficiently processed and exported to postGolgi compartments (Varga et al, 2004).…”

Improving Cell Surface Functional Expression of F508 CFTR: A Quest for Therapeutic Targets

“…This is particularly relevant to F508 CFTR as over accumulation of this mutant protein in the ER induces such a response, leading to downregulation of CFTR endogenous transcription (Rab et al, 2007;Bartoszewski et al, 2008). Aside from the above two, the inherent variation in the cellular chaperone machinery among different tissues or cell types will also significantly affect the cell surface functional expression of F508 CFTR (Varga et al, 2004;X. Wang et al, 2008;Rowe et al, 2010).…”