Biogenesis, Maturation and Surface Trafficking of Wild-Type and Mutant CFTR

Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A, I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A, I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein halflife. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr (1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.;One unusual feature of this protein is that during biogenesis, approximately 75% 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 approximately 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.;Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR ΔF508, and analysed its cell-surface trafficking after rescue [rΔF508 (rescued ΔF508) CFTR]. We show that rΔF508 CFTR endocytosis is 6-fold more rapid (∼30% per 2.5 min) than WT (wild-type, ∼5% per 2.5 min) CFTR at 37 °C in polarized airway epithelial cells (CFBE41o-). We also investigated rΔF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 °C) and following treatment with pharmacological chaperones. At low temperature, rΔF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rΔF508 CFTR is TS. Furthermore, rΔF508 CFTR is stabilized at the lower temperature; its half-life increases from 8 h at 27 °C. Pharmacological chaperone treatment at 37 °C corrected the rΔF508 CFTR internalization defect, slowing endocytosis from ∼30% per 2.5 min to ∼5% per 2.5 min, and doubled ΔF508 surface half-life from 2 to 4 h. These effects are ΔF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing ΔF508 CFTR from ERAD, but also by enhancing its cell-surface stability.