FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2070043042> ?p ?o ?g. }

• W2070043042 endingPage "8950" @default.
• W2070043042 startingPage "8942" @default.
• W2070043042 abstract "Deletion of phenylalanine at position 508 (ΔF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that ΔF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous ΔF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of ΔF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued ΔF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4–20-fold accelerated degradation rate between 37 and 40 °C is, most likely, due to decreased conformational stability of the rescued ΔF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native ΔF508 CFTR. Deletion of phenylalanine at position 508 (ΔF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that ΔF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous ΔF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of ΔF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued ΔF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4–20-fold accelerated degradation rate between 37 and 40 °C is, most likely, due to decreased conformational stability of the rescued ΔF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native ΔF508 CFTR. cystic fibrosis cystic fibrosis transmembrane conductance regulator endoplasmic reticulum brefeldin A cycloheximide endoglycosidase H peptide N-glycanase hemagglutinin monoclonal antibody transmembrane nucleotide binding domain wild type enhanced chemiluminescence sulfo-succinimidyl-2-(biotinamido)ethyl-1,2-dithiopropionate Cystic fibrosis (CF)1 is one of the most prevalent lethal genetic disorders among Caucasian populations (1Kerem B. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L.-C. Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3248) Google Scholar). The CF gene encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl− channel and conductance regulator, expressed at the apical membrane of secretory epithelia (2Rommens J.M. Iannuzzi M.C. Kerem B. Drumm M.L. Melmer G. Dean M. Rosmahel R. Cole J.L. Kennedy D. Hidaka N. Zsiga M. Buchwald M. Riordan J.R. Tsui L.-C. Collins F.S. Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2554) Google Scholar, 3Riordan J.R. Rommens J.M. Kerem B. Alon M. Rosmahel R. Grzelchak Z. Zielenski J. Lok S. Plavsic N. Chou J.-L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5976) Google Scholar). CFTR, a member of the ABC transporter family, consists of two structurally homologous halves, each comprised of six transmembrane (TM) helices and a nucleotide binding domain (NBD1 and NBD2), which are connected by the regulatory (R) domain (3Riordan J.R. Rommens J.M. Kerem B. Alon M. Rosmahel R. Grzelchak Z. Zielenski J. Lok S. Plavsic N. Chou J.-L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5976) Google Scholar). This complex, multidomain structure conceivably renders the posttranslational folding of wild type (wt) CFTR inefficient. More than 50% of the newly synthesized wt CFTR remains incompletely folded and is degraded at the endoplasmic reticulum (ER), whereas the remaining 25–50% undergoes an ATP-dependent conformational maturation and is exported to the cis/medial-Golgi, where its complex glycosylation can occur (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar, 5Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar). The hallmark of CF is the loss of cAMP-activated chloride conductance in the epithelial plasma membrane of airways, intestine, and exocrine glands (6Quinton P.M. FASEB J. 1990; 4: 2709-2717Crossref PubMed Scopus (406) Google Scholar, 7Zielenski J. Tsui L.-C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (521) Google Scholar, 8Welsh M.J. Smith A. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1235) Google Scholar). More than 900 mutations have been identified in the CF gene, leading to impaired biosynthesis, processing, activation, and/or stability of CFTR (7Zielenski J. Tsui L.-C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (521) Google Scholar, 8Welsh M.J. Smith A. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1235) Google Scholar). The most frequent mutation, deletion of phenylalanine at position 508 (ΔF508) in the NBD1, is found in >90% of the patients and detected in ∼70% of CF chromosomes (1Kerem B. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L.-C. Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3248) Google Scholar, 7Zielenski J. Tsui L.-C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (521) Google Scholar). It is believed that deletion of Phe-508 interrupts the posttranslational folding of CFTR (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar, 5Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar, 9Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 10Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 350: 761-764Crossref Scopus (1063) Google Scholar, 11Zhang F. Kartner N. Lukacs G.L. Nat. Struct. Biol. 1998; 5: 180-183Crossref PubMed Scopus (131) Google Scholar) and targets the core-glycosylated folding intermediate for degradation, predominantly via the ubiquitin-proteasome pathway at the ER (12Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-128Abstract Full Text PDF PubMed Scopus (1133) Google Scholar, 13Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (775) Google Scholar). Exposure of ER-retention signals may contribute to the inability of folding intermediate(s) to exit the ER (14Chang X.B. Cui L. Hou Y.X. Jensen T.J. Aleksandov A.A. Mengos A. Riordan J.R. Mol. Cell. 1999; 4: 137-142Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Accordingly, negligible expression of ΔF508 CFTR could be detected at the cell surface by immunochemical techniques in recombinant cells, CF primary airway cells, and CF tissues (9Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 15Denning G.M. Ostedgaard L.S. Welsh M.J. J. Cell Biol. 1992; 118: 551-559Crossref PubMed Scopus (158) Google Scholar, 16Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet. 1992; 1: 321-327Crossref PubMed Scopus (328) Google Scholar). The recognition that the ΔF508 CFTR channel is functional bothin vivo (17Pasyk E.A. Foskett J.K. J. Biol. Chem. 1995; 270: 12347-12350Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 18F. R. Drumm M.L. Wilkinson D.J. Smit L.S. Worrell R.T. Strong T.V. Frizzell R.A. Dawson D.C. Collins F.S. Science. 1991; 254: 1797-1799Crossref PubMed Scopus (422) Google Scholar, 19Dalemans W. Barbry P. Champigny G. Jallat S. Dott K. Dreyer D. Crystal R.G. Pavirani A. Lecocq J. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (572) Google Scholar) and after its reconstitution into the phospholipid bilayer (20Li C. Ramjeesingh M. Reyes E. Jensen T. Chang X. Rommens J.M. Bear C.E. Nat. Genet. 1993; 3: 311-316Crossref PubMed Scopus (155) Google Scholar) suggested that the CF phenotype could be alleviated by relocating the mutant CFTR from the ER to the plasma membrane. Reduced temperature (10Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 350: 761-764Crossref Scopus (1063) Google Scholar, 21French P.J. van Doorninck J.H. Peters R.H. Verbeek E. Ameen N.A. Marino C.R. de Jonge H.R. Bijman J. Scholte B.J. J. Clin. Invest. 1996; 98: 1304-1312Crossref PubMed Scopus (124) Google Scholar, 22Egan M.E. Schweibert E.M. Guggino W.B. Am. J. Physiol. 1995; 268: C243-C251Crossref PubMed Google Scholar), chemical chaperones (23Brown C.R. Hong-Brown L.Q. Biwersi J. Verkman A.S. Cell Stress Chaperones. 1996; 1: 117-125Crossref PubMed Scopus (362) Google Scholar, 24Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 25Bebok Z. Venglarik C.J. Panczel Z. Jilling T. Kirk K.L. Sorcher E.J. Am. J. Physiol. 1998; 275: C599-C607Crossref PubMed Google Scholar), and down-regulation of Hsp70 (26Rubenstein R.C. Zeitlin P.L. Am. J. Physiol. 2000; 278: C259-C267Crossref PubMed Google Scholar, 27Jiang C. Fang S.L. Xiao Y.F. O'Connor S.P. Nadler S.G. Lee D.W. Jefferson D.M. Kaplan J.M. Smith A.E. Cheng S.H. Am. J. Physiol. 1998; 275: C171-C178Crossref PubMed Google Scholar) activity are thought to partially revert the folding defect of ΔF508 CFTR and promote the accumulation of the functional channel at the cell surface. Importantly, using more sensitive electrophysiological techniques, constitutive accumulation of ΔF508 CFTR was documented in the plasma membrane of primary epithelia from ΔF508 homozygous mice (21French P.J. van Doorninck J.H. Peters R.H. Verbeek E. Ameen N.A. Marino C.R. de Jonge H.R. Bijman J. Scholte B.J. J. Clin. Invest. 1996; 98: 1304-1312Crossref PubMed Scopus (124) Google Scholar, 28Steagall W.K. Drumm M.L. Gastroenterology. 1999; 116: 1379-1388Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and in the intestinal and gallbladder epithelia of homozygous ΔF508 patients (29Bronsveld I. Mekus F. Bijman J. Ballman M. Greipel J. Hundrieser J. Halley D.J.J. Laabs U. Busche R. de Jonge H.R. Tummler B. Veeze H.J. Gastroenterology. 2000; 119: 32-40Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30Dray-Charier N. Paul A. Scoazec J.-Y. Veissiere D. Mergey M. Capeau J. Soubrane O. Housset C. Hepatology. 1999; 29: 1624-1634Crossref PubMed Scopus (35) Google Scholar). These studies parallel the results of recent immunolocation reports to some extent and suggest that the processing defect of the ΔF508 CFTR is tissue-specific (30Dray-Charier N. Paul A. Scoazec J.-Y. Veissiere D. Mergey M. Capeau J. Soubrane O. Housset C. Hepatology. 1999; 29: 1624-1634Crossref PubMed Scopus (35) Google Scholar, 31Kalin N. Claas A. Sommer M. Puchelle E. Tummler B. J. Clin. Invest. 1999; 103: 1379-1389Crossref PubMed Scopus (229) Google Scholar, 32Kinnman N. Lindblad A. Housset C. Buentke E. Scheynius A. Strandvik B. Hultcrantz R. Hepatology. 2000; 32: 334-339Crossref PubMed Scopus (56) Google Scholar, 33Penque D. Mendes F. Beck S. Farinha C. Pacheco P. Nogueira P. Lavinha J. Malho R. Amaral M.D. Lab. Invest. 2000; 80: 857-868Crossref PubMed Scopus (92) Google Scholar). If the ER retention of the mutant is not complete, accelerated disposal from the post-ER compartments could contribute to its inability to express at the physiological level in certain tissues. Indeed, based on indirect evidence, we proposed that the rescued ΔF508 CFTR has a short residence time at the cell surface of Chinese hamster ovary cells (CHO), expressing ΔF508 CFTR heterologously (34Lukacs G.L. Chang B.-X. Bear C. Kartner N. Mohamed A. Riordan J.R. Grinstein S. J. Biol. Chem. 1993; 268: 21592-21598Abstract Full Text PDF PubMed Google Scholar). However, neither this nor any other study has provided direct biochemical or structural information regarding the behavior of ΔF508 CFTR following its escape from ER. Here we provide direct biochemical and functional evidence for the biological instability of the complex-glycosylated ΔF508 CFTR. Furthermore, we show that the stability defect is temperature-sensitive, which is likely due to an attenuated conformational stability of the native state, demonstrated by increased protease susceptibility and the thermoaggregation tendency of the rescued mutant relative to its wt counterpart. The implications of these observations are fundamental with respect to understanding the cellular phenotype and designing more efficient therapeutic strategies in CF. A mixture of stably transfected baby hamster kidney (BHK) cells, expressing human wt and ΔF508 CFTR with a carboxyl-terminal hemagglutinin (HA) epitope, was generated and maintained as described (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar). Characterization of the HA-tagged CFTR variants will be described elsewhere. Isolation of ER, Golgi, and plasma membrane-enriched microsomes from BHK cells was performed using nitrogen cavitation and differential centrifugation as described (11Zhang F. Kartner N. Lukacs G.L. Nat. Struct. Biol. 1998; 5: 180-183Crossref PubMed Scopus (131) Google Scholar). Where specified, the core-glycosylated wt or mutant CFTR was eliminated from the cells during a 3-h incubation in the presence of cycloheximide (CHX, 100 μg/ml). The microsomal pellet was resuspended in HSE medium (10 mm sodium HEPES, 0.25 m sucrose, pH 7.6) and used either immediately or after being snap-frozen in liquid nitrogen. Microsomes were isolated from ΔF508 and wt CFTR expressor BHK cells and incubated at a protein concentration of 1.3–1.5 and 0.8–1.0 mg/ml, respectively, in the presence of trypsin or proteinase K for 15 min at 4 °C in digestion buffer (phosphate-buffered saline) as described. Proteolysis was terminated by the addition of phenylmethylsulfonyl fluoride to 1 mm, and samples were immediately denatured in 2× Laemmli sample buffer at 37 °C for 20 min. To distinguish between high mannose and complex-typeN-linked oligosaccharide modification of CFTR, cell lysates were incubated with endoglycosidase H (7 μg/ml) and peptideN-glycosidase F (31 μg/ml) at 37 °C for 3 h. The mobility shift of deglycosylated CFTR was visualized with immunoblotting using anti-HA mAb. CFTR immunoblotting was performed with anti-HA mAb (Babco). Proteolytic digestion patterns were visualized with the mouse monoclonal M3A7 and L12B4 anti-CFTR Abs. L12B4 localizes to the region of the cytoplasmic NBD1 of CFTR (epitope within the range of amino acid positions 386 and 412), and M3A7 localizes to the region of the cytoplasmic NBD2 of CFTR (epitope within the range of amino acid positions 1365 and 1395) (11Zhang F. Kartner N. Lukacs G.L. Nat. Struct. Biol. 1998; 5: 180-183Crossref PubMed Scopus (131) Google Scholar). Immunoblotting of ERP72 and GRP78 was performed with the rabbit polyclonal (SPA720) and mouse monoclonal (SPA826, Stressgen) antibodies, respectively. Immunoblots, with multiple exposures, were quantified using DuoScan transparency scanner and N.I.H. Image 6.1 software (developed by the National Institutes of Health and available at their web site). Metabolic labeling and immunoprecipitation were performed essentially as described (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar). Monolayer cells were pulse-labeled under defined conditions and chased at the indicated temperature for the time intervals specified for each experiment. Membrane proteins were solubilized in 1 ml of RIPA buffer (150 mm NaCl, 20 mm Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0) supplemented with protease inhibitors (10 μg/ml leupeptin and pepstatin, 0.5 mm phenylmethylsulfonyl fluoride, and 10 mm iodoacetamide). Immunoprecipitates, obtained with anti-HA or M3A7 and L12B4 anti-CFTR mAbs, were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. The radioactivity incorporated was quantified using a PhosphorImager (Molecular Dynamics) with ImageQuant software (Molecular Dynamics) (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar). Cells were rinsed (H-buffer: 154 mm NaCl, 10 mm HEPES, 3 mm KCl, 1 mm MgCl2, 0.1 mm CaCl2, 10 mm glucose, pH 7.6) and biotinylated with 1 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,2-dithiopropionate (EZ-Link sulfo-NHS-SS-biotin, Pierce) three times for 15 min at 37 °C. Following the solubilization of the cells in RIPA buffer, biotinylated CFTRs were affinity-isolated on streptavidin-Sepharose (Sigma), separated with SDS-polyacrylamide gel electrophoresis, and visualized with anti-HA mAb and ECL. The plasma membrane cAMP-dependent halide conductance of transfected BHK cells was determined with iodide efflux as described (35Chang B.-X. Tabcharani J.A. Hou Y.-X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). In brief, the chloride content was replaced with iodide by incubating the cells in loading buffer (136 mm NaI, 3 mm KNO3, 2 mmCa(NO3)2, 11 mm glucose, 20 mm HEPES, pH 7.4) for 60 min at room temperature. Iodide efflux was initiated by replacing the loading buffer with efflux medium (composed of 136 mm nitrate in place of iodide). The extracellular medium was replaced every minute with efflux buffer (1 ml). After a steady-state was reached, the intracellular cAMP level was raised by agonists (10 μm forskolin, 0.2 mmCTP-cAMP, and 0.2 mm isobutylmethylxanthine) to achieve maximal phosphorylation of ΔF508 CFTR, and collection of the efflux medium resumed for an additional 6–9 min. The amount of iodide in each sample was determined with an iodide-selective electrode (Orion). Following the solubilization of BHK cells in Laemmli sample buffer, the aggregation tendency of wt and mutant CFTR was compared by exposing the lysate to temperatures ranging from 37 to 100 °C for 5 min. SDS-resistant macromolecular aggregates were sedimented with centrifugation (17,000 × g for 15 min). Monomeric mutant and wt CFTR, ERP72, GRP78, and Na+/K+-ATPase remaining in the supernatant were measured with quantitative immunoblotting using the appropriate Ab and ECL. To attain high sensitivity immunodetection, the influenza HA epitope-tagged ΔF508 and wt CFTR were expressed stably in BHK cells. Immunoblot analysis showed that the HA-tagged ΔF508 CFTR appears as an endoglycosidase H (endo-H)- and peptideN-glycosidase F (PNGase-F)-sensitive, core-glycosylated polypeptide with an apparent molecular mass ≈140–150 kDa (Fig.1 a, empty arrowhead) at 37 °C (9Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). The processing defect of the ΔF508 CFTR could be partially overcome by the combination of glycerol and low temperature treatment, similarly to its nontagged counterpart, as reported in a number of cultured cells (10Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 350: 761-764Crossref Scopus (1063) Google Scholar, 23Brown C.R. Hong-Brown L.Q. Biwersi J. Verkman A.S. Cell Stress Chaperones. 1996; 1: 117-125Crossref PubMed Scopus (362) Google Scholar, 24Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Following the optimization of the rescue conditions, accumulation of the complex-glycosylated ΔF508 CFTR was indicated by the appearance of endo-H-resistant immunoreactive polypeptide with an apparent molecular mass ≈170 kDa (Fig.1 a, black arrowhead) (9Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). The N-linked oligosaccharide modification and the apparent molecular mass of the rescued ΔF508 CFTR are virtually identical to that of the HA-tagged complex-glycosylated wt CFTR (Fig. 1). The biological stability of the complex-glycosylated wt and rescued ΔF508 CFTR was determined upon inhibition of protein biosynthesis with CHX or vesicular transport from ER to Golgi with brefeldin A (BFA). After the accumulation of the complex-glycosylated ΔF508 CFTR at 26 °C, cells were incubated in the presence of CHX or BFA at 37 °C, and the remaining CFTR was measured with quantitative immunoblotting, using anti-HA mAb as a function of incubation time (Fig. 1, b and c). Densitometry revealed that the half-life of the complex-glycosylated ΔF508 CFTR (t 12 ≈ 5 h) is at least four times shorter than that of the wt CFTR (t 12 ≈ 22 h) at 37 °C, regardless of the inhibitor (Fig. 1 d). Similarly fast disposal of the ΔF508 CFTR (t 12 ≈ 5 h) could be observed in the absence of CHX or BFA, after shifting the temperature from 26 to 37 °C during the chase (Fig.1 c). To verify that the deletion of Phe-508 destabilizes the complex-glycosylated CFTR, metabolic pulse-chase experiments were performed on transfectants expressing wt or rescued ΔF508 CFTR (Fig.2 a). Phosphorimage analysis confirmed that the complex-glycosylated ΔF508 was eliminated four times faster (t 12 ≈ 4.5 h) than wt CFTR (t 12 ≈ 18 h) at 37 °C (Fig.2 b). Similar turnover rates were obtained upon rescuing the mutant with either glycerol or reduced temperature alone, and on mock-treated wt CFTR, ruling out that osmotic or cold stress can account for the difference (data not shown). The accelerated disappearance of ΔF508 CFTR also cannot be attributed to the loss of the carboxyl-terminal epitope, because similar half-lives were measured with antibodies to epitopes located in NBD1, NBD2, or at the amino-terminal tail (data not shown). Finally, preliminary results obtained on pancreatic ductal epithelia expressing the ΔF508 CFTR constitutively showed that the mutation impairs the stability of CFTR in polarized epithelia to a degree comparable with that found in nonpolarized cells (data not shown). These results collectively support the notion that the instability is an intrinsic property of the rescued ΔF508 CFTR rather than epitope- or cell-specific. To examine whether the turnover of the plasma membrane-associated mutant CFTR is similar to that of the complex-glycosylated ΔF508 CFTR pool, which comprises thetrans-Golgi network, secretory vesicles, and endosomes as well, the fate of the rescued ΔF508 CFTR was followed with cell surface biotinylation and iodide efflux measurements. The plasma membrane proteins of BHK cells, expressing ΔF508, rescued ΔF508, or wt CFTR, were covalently tagged with NHS-SS-biotin, affinity-isolated on streptavidin beads, and immunoblotted with anti-HA mAb. Both the rescued ΔF508 and the wt CFTR are amenable to biotinylation, in contrast to the ER-resident, core-glycosylated ΔF508 CFTR (Fig. 3 a). Densitometric analysis revealed that ≈50% of the biotinylated ΔF508 CFTR disappeared after 4 h and became undetectable by 10 h of chase at 37 °C (Fig. 3 b). In contrast, the turnover of biotinylated wt CFTR was more than 4-fold slower (t 12 ≈ 18 h, data not shown) than the rescued ΔF508 CFTR but comparable with the complex-glycosylated wt CFTR pool (Fig. 1 d and 2 b). The lack of endogenous cAMP-dependent anion conductance of BHK cells permitted us to monitor the arrival of functional ΔF508 CFTR to the plasma membrane by the iodide efflux assay. At permissive temperatures, the cAMP-stimulated iodide release was proportional with the length of the rescue period up to 8 h (Fig.4 a, inset). After allowing ΔF508 CFTR to accumulate at the cell surface for 5 h, raising the temperature to 37 °C evoked a rapid disappearance of the mutant. The amount of iodide released by cAMP-dependent protein kinase stimulation decreased by 50% after 4 h and became undetectable after 10 h of chase at 37 °C (Fig. 4,a and b) in cells expressing rescued ΔF508, whereas no decrease was apparent over 10 h in wt CFTR expressors. Because the cAMP-activated iodide release could not be detected in rescued mock-transfected and parental BHK cells (data not shown), the functional and the biotinylation studies jointly indicate that the ΔF508 CFTR channels are as unstable at the cell surface as in the post-ER compartments. Taken together, the immunoblot, metabolic pulse-chase, biotinylation, and iodide measurements provide the first direct evidence that the functional and biochemical half-life of the complex-glycosylated ΔF508 CFTR is 4–5-fold shorter than its wt counterpart at 37 °C, suggesting that structural differences may persist between the rescued ΔF508 and wt CFTR at the plasma membrane. To test whether the stability defect of the complex-glycosylated ΔF508 CFTR at 37 °C is related to its thermolability, pulse-labeled cells were chased at temperatures ranging from 28 to 40 °C. Although thet 12 of wt and rescued ΔF508 CFTR converged at 28 °C (≈50 h), a striking difference became apparent at temperatures above 30 °C. Rescued ΔF508 CFTR was at least 20-fold more unstable (t 12 ≈ 0.8 h) than its wt counterpart (t 12 ≈ 18 h) at 40 °C (Fig.5, a and b). In sharp contrast, no difference could be resolved in the relative turnover rates of the core-glycosylated (ER-resident) wt and ΔF508 CFTR between 28 and 40 °C (Fig. 5 b), consistent with the notion that the core-glycosylated form represents a common folding intermediate, which is less susceptible to thermal denaturation (4Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar, 5Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar,11Zhang F. Kartner N. Lukacs G.L. Nat. Struct. Biol. 1998; 5: 180-183Crossref PubMed Scopus (131) Google Scholar). Considering that unfolding of soluble and membrane proteins upon heat shock accelerates their cellular degradation (36Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1894) Google Scholar), our results suggest that the thermal resistance of the complex-glycosylated ΔF508 CFTR toward unfolding is substantially lower than its wt counterpart. Because large quantities of purified CFTR are not available to test this hypothesis directly, the protease susceptibility of native and the thermoaggregation propensity of solubilized CFTR variants were measured as indirect indicators of their structural stability. SDS-solubilized cell lysates, obtained from BHK cells expressing ΔF508, rescued ΔF508, or wt CFTR, were heat-denatured at temperatures ranging between 37 and 100 °C. Insoluble aggregates were sedimented by centrifugation, and monomeric CFTR remaining in the supernatant was quantified by immunoblotting. The thermostability of solubilized CFTR was characterized by measuring the aggregation temperature (Ta), at which 50% of monomeric CFTR is converted into SDS-resistant aggregates (Fig.6 a). The Ta of rescued ΔF508 CFTR was 10 °C lower (Ta ≈ 65 °C) than its wt counterpart (Ta ≈ 75 °C) but 10 °C higher than the core-glycosylated ΔF508 CFTR (Ta ≈ 55 °C) (Fig. 6 b). The following observations suggest that the progressively decreasing thermostability of the rescued and core-glycosylated ΔF508 CFTR is likely the consequence of structural differences. Firstly, distinct aggregation pattern was also measured on immunoprecipitated wt and mutant CFTR (data not shown), implying that their aggregation propensity is independent of other polypeptides. Secondly, no difference could be documented in the thermoaggregation of the polytopic Na+/K+-ATPase, in parental BHK cells, or in cells expressing wt, ΔF508, or rescued ΔF508 CFTR (Fig. 6 c). Conversely, ERP72 and GRP78, soluble ER proteins, were resistant to aggregation, presumably due to their fully denatured state in SDS, in contrast to polytopic membrane proteins, which tend to preserve their aggregation tendency following detergent solubilization (37Sagne C. Isambert M.F. Henry J.P. Gasnier B. Biochem. J. 1996; 316: 825-831Crossref PubMed Scopus (72) Google Scholar) (Fig. 6 c). Finally and most importantly, no significant difference between the thermoaggregation tendency of ΔF508, rescued ΔF508, and wt CFTR" @default.
• W2070043042 created "2016-06-24" @default.
• W2070043042 creator A5002738774 @default.
• W2070043042 creator A5039737636 @default.
• W2070043042 creator A5045072109 @default.
• W2070043042 creator A5052136826 @default.
• W2070043042 date "2001-03-01" @default.
• W2070043042 modified "2023-10-16" @default.
• W2070043042 title "Conformational and Temperature-sensitive Stability Defects of the ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Post-endoplasmic Reticulum Compartments" @default.
• W2070043042 cites W120703973 @default.
• W2070043042 cites W1496230634 @default.
• W2070043042 cites W1602887917 @default.
• W2070043042 cites W1606812696 @default.
• W2070043042 cites W1638694981 @default.
• W2070043042 cites W1677528798 @default.
• W2070043042 cites W1786021994 @default.
• W2070043042 cites W1901783123 @default.
• W2070043042 cites W1969697265 @default.
• W2070043042 cites W1975971473 @default.
• W2070043042 cites W1982796317 @default.
• W2070043042 cites W1989746014 @default.
• W2070043042 cites W1999746307 @default.
• W2070043042 cites W2000390486 @default.
• W2070043042 cites W2001213356 @default.
• W2070043042 cites W2005269914 @default.
• W2070043042 cites W2011259421 @default.
• W2070043042 cites W2014319545 @default.
• W2070043042 cites W2015357935 @default.
• W2070043042 cites W2026440877 @default.
• W2070043042 cites W2032258216 @default.
• W2070043042 cites W2039599894 @default.
• W2070043042 cites W2050662826 @default.
• W2070043042 cites W2052588826 @default.
• W2070043042 cites W2065444910 @default.
• W2070043042 cites W2073727172 @default.
• W2070043042 cites W2075257287 @default.
• W2070043042 cites W2078818437 @default.
• W2070043042 cites W2079234445 @default.
• W2070043042 cites W2080423498 @default.
• W2070043042 cites W2082102563 @default.
• W2070043042 cites W2088043549 @default.
• W2070043042 cites W2103634965 @default.
• W2070043042 cites W2111220371 @default.
• W2070043042 cites W2116599708 @default.
• W2070043042 cites W2117143712 @default.
• W2070043042 cites W2117148991 @default.
• W2070043042 cites W2122484880 @default.
• W2070043042 cites W2125391335 @default.
• W2070043042 cites W2125428336 @default.
• W2070043042 cites W2132775040 @default.
• W2070043042 cites W2148573587 @default.
• W2070043042 cites W2184141508 @default.
• W2070043042 cites W2187603576 @default.
• W2070043042 cites W2197268372 @default.
• W2070043042 cites W2415950711 @default.
• W2070043042 doi "https://doi.org/10.1074/jbc.m009172200" @default.
• W2070043042 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11124952" @default.
• W2070043042 hasPublicationYear "2001" @default.
• W2070043042 type Work @default.
• W2070043042 sameAs 2070043042 @default.
• W2070043042 citedByCount "203" @default.
• W2070043042 countsByYear W20700430422012 @default.
• W2070043042 countsByYear W20700430422013 @default.
• W2070043042 countsByYear W20700430422014 @default.
• W2070043042 countsByYear W20700430422015 @default.
• W2070043042 countsByYear W20700430422016 @default.
• W2070043042 countsByYear W20700430422017 @default.
• W2070043042 countsByYear W20700430422018 @default.
• W2070043042 countsByYear W20700430422019 @default.
• W2070043042 countsByYear W20700430422020 @default.
• W2070043042 countsByYear W20700430422021 @default.
• W2070043042 countsByYear W20700430422022 @default.
• W2070043042 countsByYear W20700430422023 @default.
• W2070043042 crossrefType "journal-article" @default.
• W2070043042 hasAuthorship W2070043042A5002738774 @default.
• W2070043042 hasAuthorship W2070043042A5039737636 @default.
• W2070043042 hasAuthorship W2070043042A5045072109 @default.
• W2070043042 hasAuthorship W2070043042A5052136826 @default.
• W2070043042 hasBestOaLocation W20700430421 @default.
• W2070043042 hasConcept C104317684 @default.
• W2070043042 hasConcept C12554922 @default.
• W2070043042 hasConcept C158617107 @default.
• W2070043042 hasConcept C170493617 @default.
• W2070043042 hasConcept C185592680 @default.
• W2070043042 hasConcept C24530287 @default.
• W2070043042 hasConcept C2776938444 @default.
• W2070043042 hasConcept C2777893369 @default.
• W2070043042 hasConcept C2778428886 @default.
• W2070043042 hasConcept C54355233 @default.
• W2070043042 hasConcept C55493867 @default.
• W2070043042 hasConcept C6929976 @default.
• W2070043042 hasConcept C86803240 @default.
• W2070043042 hasConcept C95444343 @default.
• W2070043042 hasConceptScore W2070043042C104317684 @default.
• W2070043042 hasConceptScore W2070043042C12554922 @default.
• W2070043042 hasConceptScore W2070043042C158617107 @default.
• W2070043042 hasConceptScore W2070043042C170493617 @default.
• W2070043042 hasConceptScore W2070043042C185592680 @default.

• next