PDZ Domain Interaction Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m206964200
ISSN1083-351X
AutoresAgnieszka Swiatecka‐Urban, Marc Duhaime, Bonita Coutermarsh, Katherine H. Karlson, James F. Collawn, Michał Milewski, Garry R. Cutting, William B. Guggino, George M. Langford, Bruce A. Stanton,
Tópico(s)Cellular transport and secretion
ResumoThe C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-ΔTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-ΔTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ∼24 to ∼13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-ΔTRL did not differ. However, endocytic recycling of CFTR-ΔTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells. The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-ΔTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-ΔTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ∼24 to ∼13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-ΔTRL did not differ. However, endocytic recycling of CFTR-ΔTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells. cystic fibrosis transmembrane conductance regulator cystic fibrosis green fluorescent protein, TGN, trans-Golgi network Madin-Darby canine kidney wild type minimum essential medium The selective expression of transport proteins in either the apical or basolateral membrane is essential for polarized epithelial cells to carry out vectorial transport of ions and water (1Brown D. Am. J. Physiol. 2000; 278: F192-F201Crossref Google Scholar, 2Brown D. Breton S. Kidney Int. 2000; 57: 816-824Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (330) Google Scholar, 4Caplan M.J. Kidney Int. 2001; 60: 427-430Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). For example, polarization of the cystic fibrosis transmembrane conductance regulator (CFTR)1 to the apical plasma membrane is required for vectorial Cl−secretion across a variety of epithelial cells including those in airway, kidney, intestine, and pancreas (4Caplan M.J. Kidney Int. 2001; 60: 427-430Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 5Stanton B.A. Wien. Klin. Wochenschr. 1997; 109: 457-464PubMed Google Scholar, 6Kunzelmann K. Rev. Physiol. Biochem. Pharmacol. 1999; 137: 1-70PubMed Google Scholar, 7Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar). In the genetic disease cystic fibrosis, the most common mutation in the CFTR gene, ΔF508, causes CFTR to fold incorrectly and to be retained in the endoplasmic reticulum (6Kunzelmann K. Rev. Physiol. Biochem. Pharmacol. 1999; 137: 1-70PubMed Google Scholar, 7Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar, 8Cheng 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 (1398) Google Scholar, 9Jilling T. Kirk K.L. Int. Rev. Cytol. 1997; 172: 193-241Crossref PubMed Google Scholar). Because ΔF508-CFTR does not reach the apical plasma membrane, epithelial cells in the airway, pancreas, and intestine do not secrete chlorine (6Kunzelmann K. Rev. Physiol. Biochem. Pharmacol. 1999; 137: 1-70PubMed Google Scholar, 7Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar, 10Denning G.M. Ostedgaard L.S. Cheng S.H. Smith A.E. Welsh M.J. J. Clin. Invest. 1992; 89: 339-349Crossref PubMed Scopus (207) Google Scholar). Transport proteins contain amino acid motifs that direct and/or localize proteins to the appropriate membrane domains (1Brown D. Am. J. Physiol. 2000; 278: F192-F201Crossref Google Scholar, 2Brown D. Breton S. Kidney Int. 2000; 57: 816-824Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (330) Google Scholar, 4Caplan M.J. Kidney Int. 2001; 60: 427-430Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Highly conserved motifs that direct the polarized expression of transport proteins to the basolateral membrane of epithelial cells include tyrosine- and dileucine-based motifs (1Brown D. Am. J. Physiol. 2000; 278: F192-F201Crossref Google Scholar, 2Brown D. Breton S. Kidney Int. 2000; 57: 816-824Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (330) Google Scholar, 4Caplan M.J. Kidney Int. 2001; 60: 427-430Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). PDZ domains, which are named for three proteins in which this domain was first described (PSD-95, Dlg, and ZO-1), also determine the polarized expression of proteins in epithelial cells and neurons (11–22). PDZ domains are modular 70–90-amino acid domains that bind to short peptide sequences at the C termini of other proteins, called PDZ interacting domains (15Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (399) Google Scholar, 21Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar, 23Fanning A.S. Anderson J.M. Curr. Top. Microbiol. Immunol. 1998; 228: 209-233PubMed Google Scholar, 24Kornau H.C. Seeburg P.H. Kennedy M.B. Curr. Opin. Neurobiol. 1997; 7: 368-373Crossref PubMed Scopus (310) Google Scholar, 25Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (271) Google Scholar). Previously, we demonstrated that a C-terminal, PDZ interacting domain is required for the polarization of CFTR to the apical plasma membrane in airway and kidney epithelial cells (16Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S.S. Mickle J.E. Milewski H. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (245) Google Scholar, 18Moyer B.D. Duhaime M. Shaw C. Denton J. Reynolds D. Karlson K.H. Pfeiffer J. Wang S.S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Biol. Chem. 2000; 275: 27069-27074Abstract Full Text Full Text PDF PubMed Google Scholar, 19Milewski M.I. Mickle J.E. Forrest J.K. Stafford D.M. Moyer B.D. Cheng J. Guggino W.B. Stanton B.A. Cutting G.R. J. Cell Sci. 2001; 114: 719-726Crossref PubMed Google Scholar). Deletion of the PDZ interacting domain (TRL, using the single letter amino acid code) abrogated the polarized expression of CFTR in the apical membrane and eliminated CFTR-mediated transepithelial Cl− secretion. However, the mechanism whereby the PDZ interacting domain directs the polarized expression of CFTR to the apical plasma membrane is unknown. The objectives of the present study were to test whether the PDZ interacting domain of CFTR is an apical membrane sorting motif that directs the trafficking of CFTR to the apical membrane or is a retention motif that results in the polarization of CFTR by selectively retaining CFTR in the apical membrane by interacting with an apical PDZ protein. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-ΔTRL were targeted to the apical and basolateral membrane domains in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical membrane sorting motif. To determine whether the PDZ interacting domain is a membrane retention motif, we measured the half-life of wt-CFTR and CFTR-ΔTRL in the apical and basolateral membranes. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ∼24 to ∼13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Deletion of the PDZ interacting domain did not affect apical membrane endocytosis of CFTR. By contrast, deleting the PDZ interacting domain decreased apical endocytic recycling of CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells. MDCK cells stably expressing GFP-CFTR fusion proteins were established and maintained in culture at 37 °C in MEM complete medium containing penicillin, streptomycin, l-glutamine, fetal bovine serum (10%), and G418 (150 μg/ml) as described previously (16Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S.S. Mickle J.E. Milewski H. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (245) Google Scholar, 26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Addition of GFP to the N terminus of CFTR had no effect on CFTR localization, trafficking, its function as a Cl− channel, or its degradation (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 27Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1749) Google Scholar). Pulse-chase and selective cell surface biotinylation studies were conducted essentially as described by Lisanti et al. (28Lisanti M.P., Le Bivic A. Sargiacomo M. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2117-2127Crossref PubMed Scopus (97) Google Scholar) to determine whether the PDZ interacting domain of CFTR is a sorting and/or a membrane retention motif. The day before pulse-chase studies G418 was removed from the cell culture medium, and sodium n-butyrate (5 mm) was added to stimulate CFTR expression as described previously (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Confluent, polarized MDCK cells grown on Transwell permeable growth supports (24-mm diameter, 0.4-μm pore size; Corning Corporation, Corning, NY; number 3412) were washed extensively in phosphate-buffered saline and incubated in complete MEM without methionine and cysteine for 60 min at 37 °C. Subsequently, the cells were metabolically labeled with Tran35S-Label Reagent (250 μCi/ml: ICN Pharmaceuticals, Inc., Cosa Mesa, CA) in complete MEM without Met and Cys for 30 min at (37 °C). The filters were washed at 4 °C in complete MEM containing an excess (10 mm) of unlabeled Met and Cys and chased for variable periods of time in complete MEM containing an excess (10 mm) of unlabeled Met and Cys at 37 °C. To measure the half-life of wt-CFTR and CFTR-ΔTRL in cell lysates, the cells were metabolically labeled with Tran35S-Label Reagent as described above, cooled to 4 °C, and lysed in lysis buffer as described previously (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). CFTR was immunoprecipitated by incubation with either a GFP polyclonal antibody (5 μg: CLONTECH, Palo Alto, CA; number 8372-2) or a mixture of monoclonal antibodies M3A7 and L12B4A (29Chang X.B. Kartner N. Seibert F.S. Aleksandrov A.A. Kloser A.W. Kiser G.L. Riordan J.R. Methods Enzymol. 1998; 292: 616-629Crossref PubMed Scopus (15) Google Scholar) (2 μg each; Upstate Biotechnology, Inc., Waltham, MA) followed by a second incubation with protein A or protein G (as appropriate) conjugated to Sepharose beads (Pierce). Immunoprecipitated CFTR was eluted from the protein A- or G-Sepharose beads by incubation at 95 °C for 5 min in SDS sample buffer and centrifuged for 1 min at 14,000 ×g. Immunoprecipitated and 35S-labeled wt-CFTR and CFTR-ΔTRL were separated by 7.5% SDS-PAGE. To test the hypothesis that the PDZ interacting domain is a sorting motif, the cells were pulse-labeled with Tran35S-Label Reagent (Met/Cys: 250 μCi/ml) for 30 min, chased for 0, 30, 60, 90, or 120 min, and then cooled to 4 °C. The proteins in the apical and basolateral membranes were selectively biotinylated (EZ-Link™ Biotin-LC-Hydrazide; Pierce) as described in detail previously (16Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S.S. Mickle J.E. Milewski H. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (245) Google Scholar,26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Subsequently, the cells were lysed, biotinylated proteins were isolated by streptavidin beads, and biotinylated CFTR was immunoprecipitated using a GFP monoclonal antibody (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) or a mixture of M3A7/L12B4 monoclonal antibodies (29Chang X.B. Kartner N. Seibert F.S. Aleksandrov A.A. Kloser A.W. Kiser G.L. Riordan J.R. Methods Enzymol. 1998; 292: 616-629Crossref PubMed Scopus (15) Google Scholar). Immunoprecipitated and biotinylated 35S-labeled wt-CFTR and CFTR-ΔTRL were separated by 7.5% SDS-PAGE. Detection of 35S-labeled wt-CFTR and CFTR-ΔTRL was conducted by placing gels on a general purpose storage phosphorus screen (Molecular Dynamics, Sunnyvale, CA) and, 24–72 h later, detection using the Storm 860 PhosphorImager system (Molecular Dynamics). Measurement of 35S-labeled wt-CFTR and CFTR-ΔTRL was conducted using a Dell OptiPlex GX1 computer and ImageQuant 5.1 software (Molecular Dynamics). Studies were conducted to determine whether apical polarization of CFTR was mediated in part by transcytosis of CFTR from the basolateral to the apical plasma membrane according to a method described in detail previously (30Le Bivic A. Real F.X. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9313-9937Crossref PubMed Scopus (145) Google Scholar). In brief, basolateral membrane proteins were biotinylated at 4 °C using a derivative of biotin (EZ-Link™ Sulfo-NHS-SS-Biotin: Pierce) that can be reduced by GSH. Subsequently, the cells were warmed to 37 °C for 0, 60, or 120 min, and the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins in the apical or basolateral membranes were reduced by GSH added to the apical or basolateral solutions, respectively, for 30 min at 4 °C. In preliminary studies we demonstrated that GSH only reduces the disulfide bonds of biotinylated proteins in plasma membranes and does not cross membranes or monolayers of polarized MDCK cells. Biotinylated CFTR was analyzed by Western blotting using a GFP monoclonal antibody (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) or a mixture of M3A7/L12B4 monoclonal antibodies (29Chang X.B. Kartner N. Seibert F.S. Aleksandrov A.A. Kloser A.W. Kiser G.L. Riordan J.R. Methods Enzymol. 1998; 292: 616-629Crossref PubMed Scopus (15) Google Scholar) and an anti-mouse horseradish peroxidase antibody using the Western Lightning™ Chemiluminescence Reagent Plus detection system (ECL). Studies were conducted to determine whether the reduced half-life of CFTR-ΔTRL compared with wt-CFTR in the apical membrane was due to a difference in endocytosis according to a method described in detail previously (26Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 31Weixel K. Bradbury N.A. Methods Mol. Med. 2002; 70: 323-340PubMed Google Scholar). In brief, apical membrane proteins were biotinylated at 4 °C using EZ-LinkTM Sulfo-NHS-SS-Biotin (Pierce). Subsequently, the cells were warmed to 37 °C for 1, 3, 5, or 10 min, and the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by GSH added to the apical solution for a total of 90 min at 4 °C. At this point in the protocol, biotinylated proteins reside within the endosomal compartment. Subsequently, the cells were lysed, and the biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. Biotinylated CFTR was analyzed by Western blot analysis as described above. Studies were conducted to determine whether the reduced half-life of CFTR-ΔTRL compared with wt-CFTR in the apical membrane was due to a difference in endocytic recycling according to a method described in detail previously (32Watts C. Davidson H.W. EMBO J. 1988; 7: 1937-1945Crossref PubMed Scopus (62) Google Scholar,33Garza L.A. Birnbaum M.J. J. Biol. Chem. 2000; 275: 2560-2567Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, apical membranes were biotinylated at 4 °C and then warmed to 37 °C for 3 min to load endocytic vesicles with biotinylated proteins, including CFTR. Subsequently, the cells were cooled to 4 °C, and the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins in the apical membranes were reduced by GSH, as described for the endocytic assay. Subsequently, the cells were either lysed or warmed again to 37 °C for 3 or 5 min (to allow internalized, biotinylated CFTR to recycle to the apical membrane). The cells were then cooled again to 4 °C, the disulfide bonds on Sulfo-NHS-SS-biotinylated proteins in the apical membranes were reduced with GSH, and the cells were lysed. The biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. Biotinylated CFTR was analyzed by Western blot analysis as described above. Endocytic recycling of CFTR was expressed as the difference between the amount of biotinylated CFTR after the first and second warming to 37 °C. Biotinylated CFTR was analyzed as described above. Each experiment was repeated a minimum of three to six times. In each experiment three to six filters were studied at each time point. Calculation of the half-life of CFTR was performed using GraphPad Prism version 3.0a for Macintosh (GraphPad Software, San Diego, CA). Statistical analysis of the data was performed using GraphPad Instat version 3.0a for Macintosh (GraphPad Software). The means were compared by the unpaired t test. Ap value of <0.05 was considered significant. The data are expressed as the means ± S.E. To test the hypothesis that the PDZ interacting domain (TRL) is a sorting motif, studies were conducted in MDCK cells using a pulse-chase and domain-specific cell surface biotinylation protocol. MDCK cells stably expressing GFP-wt-CFTR or GFP-CFTR-ΔTRL were washed in a Met- and Cys-free MEM solution for 60 min, then pulsed with Tran35S-labeled Met/Cys for 30 min to label newly synthesized proteins, and chased for 0, 30, 60, 90, and 120 min in MEM containing an excess of unlabeled Cys/Met. Subsequently, apical and basolateral membrane proteins were selectively biotinylated at 4 °C, the cells were lysed, and the biotinylated proteins were isolated by streptavidin beads. Biotinylated CFTR was immunoprecipitated using either a GFP monoclonal antibody or a mixture of M3A7/L12B4 monoclonal antibodies. Similar results were obtained using either the GFP or the M3A7/L12B4 antibodies. 35S-Labeled and biotinylated CFTR was separated by SDS-PAGE and detected by PhosphorImager analysis (see “Experimental Procedures” for details). If the PDZ interacting domain is an apical membrane-sorting motif, newly synthesized wt-CFTR should be sorted directly from the trans-Golgi network (TGN) to the apical membrane with little or no wt-CFTR appearing in the basolateral membrane. By contrast, CFTR-ΔTRL should be delivered to the apical and basolateral membranes in equal amounts at each time point. Newly synthesized wt-CFTR appeared in the apical and basolateral membranes at the same rate at 0, 30, 60, and 90 min into the chase period (Fig. 1 A). The ratio of wt-CFTR in the apical/basolateral membrane was ∼1 at all time points between 0 and 90 min into the chase period (Fig.2). Thereafter (120 min) wt-CFTR accumulated preferentially in the apical plasma membrane. At 120 min into the chase period, the ratio of wt-CFTR in the apical to basolateral membrane was ∼2 (Figs. 1 A and 2). Similar results were obtained in pulse-chase studies on wt-CFTR in Calu-3 cells, a human airway epithelial cell line expressing native wt-CFTR (data will be presented in a separate study). These data suggest that the polarized expression of wt-CFTR to the apical membrane in the steady state does not result from the selective sorting of wt-CFTR from the TGN directly to the apical plasma membrane.Figure 2Summary of the ratio of wt-CFTR and CFTR -ΔTRL in the apical/basolateral membranes as a function of time after the end of the 30 min pulse with Tran35S-labeled Met/Cys. The data are expressed as the means ± S.E. where the number of experiments was four for wt-CFTR and five for CFTR-ΔTRL. The ratio of wt-CFTR in the apical/basolateral membrane was significantly different from unity at the 120-min time point, as indicated by the asterisk(p < 0.01). By contrast, the ratio of CFTR-ΔTRL in the apical/basolateral membrane was not different from unity at any time point.View Large Image Figure ViewerDownload (PPT) Similar pulse-chase and selective cell surface biotinylation studies were conducted in MDCK cells stably expressing CFTR-ΔTRL, which does not polarize in the steady state to the apical or basolateral membranes (16Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S.S. Mickle J.E. Milewski H. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (245) Google Scholar, 18Moyer B.D. Duhaime M. Shaw C. Denton J. Reynolds D. Karlson K.H. Pfeiffer J. Wang S.S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Biol. Chem. 2000; 275: 27069-27074Abstract Full Text Full Text PDF PubMed Google Scholar). Newly synthesized CFTR-ΔTRL appeared in the apical and basolateral membranes at the same rate at 0, 30, 60, 90, and 120 min into the chase period (Fig. 1 B). The ratio of CFTR-ΔTRL in the apical/basolateral membrane was ∼1 at all time points between 0 and 120 min into the chase period (Fig. 2). Taken together, our studies with wt-CFTR and CFTR-ΔTRL indicate that the PDZ interacting domain is not an apical membrane sorting motif. Thus, the polarized expression of wt-CFTR in the apical membrane of MDCK cells in the steady state does not result from the selective sorting of wt-CFTR from the TGN directly to the apical plasma membrane. Additional studies were conducted to test the hypothesis that the PDZ interacting domain is an apical membrane retention motif and that selective retention in the apical membrane of wt-CFTR leads to its apical polarization in the steady state. According to this hypothesis, the half-life of wt-CFTR in the apical membrane should be greater that its half-life in the basolateral plasma membrane. Moreover, deletion of the PDZ interacting domain should reduce the half-life of CFTR in the apical but not in the basolateral membrane. To test this hypothesis, pulse-chase and domain-specific cell surface biotinylation studies were conducted as described above, except that the chase periods were 2, 6, 18, and 24 h. As illustrated in Figs. 3 and4, the half-life of wt-CFTR in the apical membrane (24.1 h) was significantly longer than the half-life of wt-CFTR in the basolateral membrane (12.9 h). Deletion of the PDZ interacting domain significantly reduced the half-life of CFTR in the apical membrane from 24.1 to 12.6 h. By contrast, deletion of the PDZ interacting domain had no effect on the half-life of CFTR in the basolateral membrane (12.9 h for wt-CFTR and 11.2 h for CFTR-ΔTRL). Taken together, these data are consistent with the view that the PDZ interacting domain in CFTR is an apical membrane retention motif and that wt-CFTR is selectively retained in the apical membrane via interaction with a PDZ protein(s).Figure 4Summary of studies conducted to determine the half-life of wt-CFTR and CFTR -ΔTRL in the apical and basolateral membranes. The data are reported as the percentages of wt-CFTR and CFTR-ΔTRL remaining in the apical and basolateral membrane as a function time after the end of the 30-min pulse with Tran35S-labeled Met/Cys. Based on half-life of CFTR in the apical (24.1 h) and basolateral membrane (12.9 h), we estimate that, at steady state, the ratio of CFTR in the apical/basolateral membrane should be ∼2:1. This value is similar to the range of values measured for wt-CFTR previously in MDCK and human airway epithelial cells (i.e. 3:1 to 8:1) (16Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S.S. Mickle J.E. Milewski H. Cutting G.R. Guggino W.B., Li, M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (245) Google Scholar)). Moreover, the value of 2:1 is similar to the apical/basolateral ratio of a GFP-tagged fusion protein (3:1) recently reported in MDCK cells by Simons and co-workers (37Keller P. Toomre D. Diaz E. White J. Simons K. Nat. Cell Biol. 2001; 3: 140-149Crossref PubMed Scopus (367) Google Scholar). The data are expressed as the means ± S.E. where the number of experiments was six for wt-CFTR and CFTR-ΔTRL.View Large Image Figure ViewerDownload (PPT) To examine the role of the PDZ interacting domain in the degradation and stability of CFTR in the intracellular compartment(s), pulse-chase studies were conducted, essentially as described above, except that CFTR was immunoprecipitated from cell lysates after biotinylated proteins had been removed by streptavidin isolation. Thus, nonbiotinylated CFTR was immunoprecipitated and separated by SDS-PAGE. The half-life of maturely glycosylated (C band) wt-CFTR was 12.5 h, and the half-life of maturely glycosylated CFTR-ΔTRL was 11.9 h (Fig. 5). These results confirmed previous studies demonstrating that short truncations of the C terminus of CFTR (<26 amino acids) have no effect on the degradation of the maturely glycosylated (C band) CFTR (34Haardt M. Benharouga M. Lechardeur D. Kartner N. Lukacs G.L. J. Biol. Chem. 1999; 274: 21873-21877Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 35Benharouga M. Haardt M. Kartner N. Lukacs G.L. J. Cell Biol. 2001; 153: 957-970Crossref PubMed Scopus (74) Google Scholar). The data presented above do not rule out the possibility that apical membrane polarization of wt-CFTR results in part from the transcytosis of wt-CFTR from the basolateral to the apical membrane. Transcytosis of wt-CFTR may be a mechanism that, in
Referência(s)