Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal
1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês
10.1074/jbc.274.6.3602
ISSN1083-351X
AutoresLawrence S. Prince, Krisztina Peter, Sean R. Hatton, Lolita Zaliauskiene, Laura Cotlin, John P. Clancy, Richard B. Marchase, James F. Collawn,
Tópico(s)Cellular transport and secretion
ResumoWe previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192–5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2–78) and carboxyl-terminal tail regions (residues 1391–1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2–78) and CFTR-(1391–1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440–1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had ∼40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis. We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192–5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2–78) and carboxyl-terminal tail regions (residues 1391–1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2–78) and CFTR-(1391–1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440–1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had ∼40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis. Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; TR, transferrin receptor; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; BSA, bovine serum albumin; PBS, phosphate-buffered saline. (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar), which functions as a chloride channel on the apical surface of epithelial cells (2Anderson M.P. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Science. 1991; 251: 679-682Crossref PubMed Scopus (433) Google Scholar, 3Bear C.E. Li C. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (779) Google Scholar). The most common mutation in CF, ΔF508, is a temperature-sensitive mutant that fails to exit the endoplasmic reticulum, presumably because of a protein folding defect (4Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1063) Google Scholar). Previous studies have demonstrated that CFTR is endocytosed (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar, 6Lukacs G.L. Segal G. Kartner N. Grinstein S. Zhang F. Biochem. J. 1997; 328: 353-361Crossref PubMed Scopus (124) Google Scholar) through clathrin-coated vesicles (6Lukacs G.L. Segal G. Kartner N. Grinstein S. Zhang F. Biochem. J. 1997; 328: 353-361Crossref PubMed Scopus (124) Google Scholar, 7Bradbury N.A. Cohn J.A. Venglarik C.J. Bridges R.J. J. Biol. Chem. 1994; 269: 8296-8302Abstract Full Text PDF PubMed Google Scholar), suggesting that CFTR internalization may provide a mechanism for controlling the cAMP-stimulated chloride channel activity at the cell surface (6Lukacs G.L. Segal G. Kartner N. Grinstein S. Zhang F. Biochem. J. 1997; 328: 353-361Crossref PubMed Scopus (124) Google Scholar). Others have suggested that CFTR may play additional roles by regulating plasma membrane recycling (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar, 8Bradbury N.A. Jilling T. Berta G. Sorscher E.J. Bridges R.J. Kirk K.L. Science. 1992; 256: 530-532Crossref PubMed Scopus (298) Google Scholar) and in clearance of Pseudomonas aeruginosa from the respiratory tract (9Pier G.B. Grout M. Zaidi T.S. Olsen J.C. Johnson L.G. Yankaskas J.R. Goldberg J.B. Science. 1996; 271: 64-67Crossref PubMed Scopus (368) Google Scholar, 10Pier G.B. Grout M. Zaidi T.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12088-12093Crossref PubMed Scopus (278) Google Scholar). The purpose of this study was to determine the structural features of the CFTR protein required for internalization. Internalization signals identified to date include tyrosine-based motifs (YXXφ or NPXY, where X is any amino acid and φ is a bulky hydrophobic residue), dileucine motifs, and acidic cluster/casein kinase II-based motifs (11Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 12Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar, 13Letourneur F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 14Voorhees P. Deignan E. Donselaar E.V. Humphrey J. Marks M.S. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar, 15Mauxion F. Le Borgne R. Munier-Lehmann H. Hoflack B. J. Biol. Chem. 1996; 271: 2171-2178Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Initial studies of type III membrane proteins indicate that the targeting signals occur in the amino- and carboxyl-terminal cytoplasmic tail regions (16Piper R.C. Tai C. Kulesza P. Pang S. Warnock D. Baenziger J. Slot J.W. Geuze H.J. Puri C. James D.E. J. Cell Biol. 1993; 121: 1221-1232Crossref PubMed Scopus (97) Google Scholar, 17Corvera S. Chawla A. Chakrabarti R. Joly M. Buxton J. Czech M.P. J. Cell Biol. 1994; 126: 979-989Crossref PubMed Scopus (103) Google Scholar, 18Garippa R.J. Judge T.W. James D.E. McGraw T.E. J. Cell Biol. 1994; 124: 705-715Crossref PubMed Scopus (70) Google Scholar, 19Tan P.K. Waites C. Liu Y. Krantz D.E. Edwards R.H. J. Biol. Chem. 1998; 273: 17351-17360Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Our initial studies on the identification of CFTR internalization signals focused on the two tail regions of the CFTR molecule. Here we show that both the amino- and carboxyl-terminal cytoplasmic tail regions of CFTR, residues 2–78 and 1391–1476, are individually sufficient to promote rapid internalization of a reporter molecule, the transferrin receptor (TR). We also demonstrate both in the context of chimeric and native proteins that tyrosine 1424 is important for CFTR endocytosis. Furthermore, we show that the intracellular distribution of the CFTR-TR chimeras is similar to that of the TR, suggesting that endocytosis may regulate CFTR activity at the cell surface. The CFTR-TR chimeras were constructed using the polymerase chain reaction as described previously (20Collawn J.F. Lai A. Domingo D. Fitch M. Hatton S. Trowbridge I.S. J. Biol. Chem. 1993; 268: 21686-21692Abstract Full Text PDF PubMed Google Scholar). A polymerase chain reaction was performed on pKCTR-CFTR cDNA (also referred to as pGT-CFTR), and unique NheI andAflII sites were introduced in the 5′ and 3′ primers, respectively. For the amino-terminal CFTR tail, the 5′ and 3′ primers were 5′-AA-GCT-AGC-CAG-AGG-TCG-CCT-CTG-GAA-AA-3′ and 5′-AA-CTT-AAG-GAA-AAAACA-TCG-CCG-AAG-GGC, respectively. For the carboxyl-terminal CFTR tail, the 5′ and 3′ primers were 5′-GCT-AGC-GCA-TTT-GCT-GAT-TGC-ACA-GTA-ATT-3′ and 5′-CTT-AAG-TTG-CAC-CTC-TTC-TTCTGT-CTC-CTC-3′, respectively. The polymerase chain reaction-generated fragment was then subcloned into pBluescript SK+ with a human TR insert containing these two sites (ATG-ATG-GCT-AGC-CTT-AAG-AGG) encoding a seven-residue cytoplasmic tail with the sequence Met-Met-Ala-Ser-Leu-Lys-Arg (21Lai A. Sisodia S.S. Trowbridge I.S. J. Biol. Chem. 1995; 270: 3565-3573Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The addition of the two restriction sites adds three residues, Ala-Ser-Leu, to the tailless TR (Δ3–59; Ref. 22Jing S. Spencer T. Miller K. Hopkins C. Trowbridge I.S. J. Cell Biol. 1990; 110: 283-294Crossref PubMed Scopus (220) Google Scholar), and the amino- and carboxyl-terminal regions of CFTR were inserted between the Ser and Leu residues (Fig. 1). Mutations were introduced into the amino- and carboxyl-terminal tail regions using the Chameleon double-stranded, site-directed mutagenesis kit (Strategene). The mutations were verified by dideoxynucleotide sequencing (23Sanger F. Nicklen S. Coulson R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar, 24Tabor S. Richardson C.C. Biochem. J. 1987; 84: 4767-4771Google Scholar) of the BH-RCAS constructs using the Sequenase kit (U.S. Biochemical Corp.). Human TR and CFTR-TR chimeras were expressed in chicken embryo fibroblasts as described previously (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Crossref PubMed Scopus (160) Google Scholar) using the BH-RCAS expression vector (26Hughes S.H. Greenhouse J.J. Petropoulos C.J. Sutrave P. J. Virol. 1987; 61: 3004-3012Crossref PubMed Google Scholar, 27Hughes S.H. Petropoulos C.J. Federspiel M.J. Sutrave P. Forry-Schaudies S. Bradac J.A. J. Reprod. Fertil. 1990; 41 (Suppl.): 39-49Google Scholar). The rate of transferrin internalization was determined using the IN/SUR method (28Wiley H.S. Cunningham D.D. J. Biol. Chem. 1982; 257: 4222-4229Abstract Full Text PDF PubMed Google Scholar) as described previously (29Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). pMT-CFTR (wild type) and pMT-CFTR-A1440X were kindly provided by Dr. Seng Cheng (Genzyme) (30Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.L. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (537) Google Scholar). pKCTR-CFTR (wild type) was provided by Dr. Eric Sorscher and the Gregory James Cystic Fibrosis Research Center Vector Core (University of Alabama at Birmingham) (31Logan J.J. Bebok Z. Walker L.C. Peng S. Felgner P.L. Giegal G.P. Frizzell R.A. Dong J. Howard M. Matalon S. Lindsey J.R. DuVall M. Sorscher E.J. Gene Ther. 1995; 2: 38-49PubMed Google Scholar). For mutagenesis of the amino-terminal region of CFTR, a XmaI–XbaI fragment of pKCTR-CFTR was subcloned into pSK-Bluescript (Stratagene). For mutagenesis of the carboxyl-terminal region of CFTR, aBstXI–SgrAI fragment from pKCTR-CFTR was subcloned into pSK-Bluescript. CFTR point mutations or deletions in the amino- or carboxyl-terminal tail regions were prepared from the corresponding pSK-Bluescript vectors (containing either theXmaI–XbaI or BstXI–SgrAI fragments, respectively) from single-stranded DNA as described previously (11Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar) by the method of Kunkel (32Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). Mutants were selected by restriction mapping or sequencing and then subcloned into theXmaI–XbaI or BstXI–SgrAI site of pKCTR-CFTR. The mutations were verified by dideoxynucleotide sequencing (24Tabor S. Richardson C.C. Biochem. J. 1987; 84: 4767-4771Google Scholar) using the Sequenase kit (U.S. Biochemical Corp.) according to the manufacturer's directions. Transient expression of wild type or mutant CFTR in COS-7 cells was performed as described by Cheng et al. (30Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.L. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (537) Google Scholar). The transfected cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and incubated at 37 °C in humidified air with 5% CO2 for 48 h. CFTR function in individual cells was assayed using the halide-quenched dye SPQ (30Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.L. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (537) Google Scholar). Briefly, cells were loaded for 10 min with SPQ (10 mm) by hypotonic shock and then mounted in a specially designed perfusion chamber for fluorescence measurements. Fluorescence (F) of single cells was measured with a Zeiss inverted microscope, a PTI imaging system, and Hamamatsu camera. Excitation was at 340 nm, and emission was >410 nm. All functional studies were at 37 °C. At the beginning of the experiments, cells were bathed in a quenching buffer (NaI buffer; 130 mm NaI, 5 mm KNO3, 2.5 mmCa(NO3)2, 2.5 mmMg(NO3)2, 10 mmd-glucose, 10 mm HEPES), and following establishment of a stable base line, they were switched to a halide-free (NO3) dequenching buffer at 200 s. Cells were stimulated with agonist unless otherwise indicated at 500 s and then returned to the quenching NaI buffer. Fluorescence was normalized to the base-line (quenched) value (average fluorescence from 100 to 200 s), with increases presented as percentage of increaseF over basal level. Each curve was generated from the mean values (±S.E.) of either 1) responding cells (defined by increase in dequench slope of >100% following cAMP stimulation (Fig.8 A wt CFTR, and Fig. 8 B, wt CFTR and 1440X CFTR)), or 2) the total number of screened cells expressing a given construct. Thenumbers in parentheses in the keys are the total cells studied (denominator) and the number of responding cells (numerator). Statistical analysis was by χ2 testing, comparing the number of responding cells in each condition with the wild-type CFTR (Fig. 8 A) or the mock condition (Fig. 8 B). The buffers used in the SPQ assay were 1) NaI buffer, pH 7.3, and 2) NaNO3 buffer (identical to NaI buffer except that 130 mm NaNO3 replaces NaI). Cell surface CFTR biotinylation was performed as described previously (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). Biotinylated and nonbiotinylated proteins were separated on an immobilized monomeric avidin column (Pierce) as described previously (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). The unbound fraction and biotin eluent fraction were then quantitated as described below. Wild-type or mutant CFTRs were immunoprecipitated from the pooled fractions (either unbound fraction or biotin eluent fraction) with either anti-C-terminal (24-1) or anti-R domain (13-1) monoclonal antibodies generously supplied by Dr. Seng Cheng (Genzyme). The CFTR immunoprecipitates were phosphorylated with [γ-32P]ATP and cAMP-dependent protein kinase and then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described previously (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). Quantitation was performed using a Molecular Dynamics PhosphorImager. The percentage of cell surface CFTR was calculated by dividing the number of counts/min detected in the CFTR C-band of the biotin eluent fraction by the total amount of CFTR C-band found in both the unbound and biotin eluent fractions (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). COS-7 cells expressing wild-type or mutant CFTRs were trypsinized 12 h after transfection and plated onto glass coverslips. 48 h later, the coverslips were fixed in methanol/acetic acid (3:1) for 30 min at −20 °C, rinsed with 1% BSA in PBS for 10 min at room temperature, and incubated with either monoclonal antibody 24–1 or 13–1 (diluted 1:50 in PBS/BSA) for 1 h at room temperature. Coverslips were then incubated for 5 min in 1% BSA in PBS (three times), incubated with Texas Red-conjugated goat anti-mouse antibody (diluted 1:1000 in PBS, 1% BSA, 5% normal goat serum) for 1 h at room temperature, and then rinsed for 5 min in 1% BSA in PBS (3 times). Coverslips were mounted in 1 mg/mlp-phenylenediamine in a 1:10 mixture of PBS/glycerol and sealed with nail polish. Slides were analyzed o a Leitz fluorescence microscope equipped with a Vario Orthomat II camera system and a Texas Red epifluorescence filter module. Micrographs were prepared on T-Max 400 film processed at ASA 800. Chicken embryo fibroblasts expressing the CFTR-TR chimeras and wild-type TR were plated onto glass coverslips and cultured overnight. The coverslips were then rinsed with PBS and fixed in 2% formaldehyde in PBS for 15 min at room temperature; rinsed with PBS; quenched with 0.37% glycine, 0.27% NH4Cl in PBS; and permeabilized with PHS (PBS, 10% horse serum, 0.1% saponin) for 30 min at room temperature. The coverslips were then incubated with JS8 mouse anti-chicken TR antibody (diluted in PHS) and with rabbit anti-human TR (1:500 in PHS) for 30 min at 37 °C and rinsed in PHS at room temperature. The coverslips were next incubated with Texas Red-labeled goat anti-mouse IgG1 (1:50 dilution in PHS) and with Oregon Green-labeled goat anti-rabbit antibody (1:50 in PHS) for 30 min at 37 °C and then rinsed, mounted, and analyzed as described above. To identify the regions of CFTR important for endocytosis, we prepared chimeric molecules consisting of the amino terminus (residues 2–78) and the carboxyl terminus of CFTR (residues 1391–1476), each fused to the transmembrane and extracellular domains of the human TR (Fig. 1). Both chimeras along with a wild-type TR and Δ3–59 TR (a mutant TR that is very poorly internalized (11Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar)) were expressed in chicken embryo fibroblasts using BH-RCAS, a replication-competent retroviral vector derived from the Rous sarcoma virus (26Hughes S.H. Greenhouse J.J. Petropoulos C.J. Sutrave P. J. Virol. 1987; 61: 3004-3012Crossref PubMed Google Scholar, 27Hughes S.H. Petropoulos C.J. Federspiel M.J. Sutrave P. Forry-Schaudies S. Bradac J.A. J. Reprod. Fertil. 1990; 41 (Suppl.): 39-49Google Scholar). Cell surface expression of both chimeras was confirmed using indirect immunofluorescence and125I-labeled transferrin binding at 4 °C (data not shown). Internalization rates of the CFTR-TR chimeras were monitored using the IN/SUR method of Wiley and Cunningham (28Wiley H.S. Cunningham D.D. J. Biol. Chem. 1982; 257: 4222-4229Abstract Full Text PDF PubMed Google Scholar). Analysis of residue 2–78 CFTR-TR and residue 1391–1476 CFTR-TR indicated that both were internalized rapidly (k e = 0.126 and 0.061, respectively, similar in fact to the wild-type TR (k e = 0.090; Fig.2 A). For comparison, the Δ3–59 TR lacking an internalization signal (11Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar) was internalized very slowly (k e = 0.009). This suggested that both cytoplasmic tail regions of CFTR were sufficient to promote TR endocytosis. To determine if the potential acidic cluster/casein kinase II region of CFTR (residues 1469–1474) in the carboxyl-terminal tail was important for endocytosis, we prepared a deletion mutant lacking this region, CFTR-(1391–1440) (Fig. 1) and compared the internalization rates of the two chimeras. The results indicate that CFTR-(1391–1440) and CFTR-(1391–1476) chimeras were internalized with similar kinetics (k e = 0.063 versus0.061, respectively; Fig. 2 A), suggesting that the membrane-distal portion of the carboxyl-terminal tail of CFTR was not required for efficient endocytosis. Next, we analyzed CFTR-TR chimeras that contained point mutations in potential internalization signals in both cytoplasmic tail regions: Y38A, L69A, Y1424A, and L1430A (Fig. 1). Analysis of these mutants in internalization assays indicated that only one mutation, Y1424A, affected the internalization rate of the chimeras (Fig. 2 B; ∼40% loss of internalization activity, p < 0.05), suggesting that this residue might be a part of an internalization signal. Since overexpression of the human wild type transferrin receptor (up to 10-fold over the endogenous receptor) does not affect internalization of the endogenous receptor in this cell type (not shown), the endocytic machinery should not be limiting for analysis of the CFTR chimeras. Therefore, we were unable to determine if the CFTR chimeras competed for the same cytosolic factors as the endogenous TR receptors. To examine the intracellular distribution of the chimeras, we compared their distribution to that of the endogenous transferrin receptor using immunofluorescence microscopy. CFTR-(2–78) was localized predominantly to juxtanuclear structures that largely co-localized with the native chicken TR receptor (Fig. 3 B). This is similar to co-localization of the human TR expressed in these cells compared with the endogenous receptor (Fig. 3 A) except that the relative surface expression of the wild-type TR appeared to be much higher (shown in green) than that of CFTR-(2–78) chimera. Very few vesicles were CFTR-(2–78)- (Fig. 3 A) or CFTR-(1391–1440)- (Fig. 3 B) positive only, but there were vesicles containing only TR (shown in red). The intracellular distribution of the CFTR-(1391–1440) chimera (Fig.3 C) appeared similar to that of the CFTR-(2–78) chimera but very different from a "tailless" TR (Δ3–59 TR) that lacks intracellular sorting signals and is localized primarily to the cell surface (Fig. 3 D). Comparison of the CFTR-(1391–1440) with the Y1424A mutant showed little or no difference (not shown). In nonpermeabilized cells, the wild type and tailless TRs are strongly positive by immunofluorescence, whereas the two CFTR chimeras are only weakly positive (not shown). 125I-Labeled transferrin binding at 4 °C indicated that the relative surface expression of both CFTR chimeras is approximately 5–10-fold lower than the wild-type TR (not shown). These results suggest that both chimeras co-localized with the endogenous TR and therefore were a part of the constitutive recycling pathway as is the case for the native TR. Having established that both cytoplasmic tail regions were sufficient for endocytosis, we next determined if they were necessary in the context of the CFTR protein. CFTR, unlike the TR, is a type III membrane protein with 12 membrane-spanning domains. In addition to the R-domain and the two nucleotide binding domains, both amino- and carboxyl-terminal tails of CFTR are cytoplasmic in orientation (Fig.4). Using a cell surface two-step biotinylation assay to monitor CFTR endocytosis (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar) that relies on biotinylation of the carbohydrate side chains found in extracellular loop 4 (Fig. 4), we compared the internalization rate of wild-type CFTR to a previously described premature stop mutant, A1440X(33Rich D.P. Gregory R.J. Cheng S.H. Smith A.E. Welsh M.J. Receptors Channels. 1993; 1: 221-232PubMed Google Scholar). First, we confirmed that A1440X expressed in COS-7 cells is maturely glycosylated by monitoring band C formation (Fig.5 A). Next, using the surface biotinylation assay, we monitored CFTR and A1440X clearance from the cell surface (Fig.6 A). Interestingly, the A1440X premature stop mutant was internalized faster than the wild-type CFTR, suggesting, as had been seen for the chimeric protein, that the last 41 residues in CFTR were not necessary for rapid endocytosis.Figure 5Analysis of CFTR and CFTR mutants expressed in COS-7 cells. CFTR and CFTR mutants expressed in COS-7 cells were immunoprecipitated from cell lysates 48 h posttransfection. The immunoprecipitated CFTR or CFTR mutants were then in vitro phosphorylated with protein kinase A and [γ-32P]ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described previously (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). The positions of bands B and C are indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Comparisons of the internalization rates of CFTR and CFTR mutants. A, internalization of CFTR and A1440X in COS-7 cells. COS-7 cells transfected with wild-type CFTR (pMT-CFTR) or A1440X (pMT-1440X) were analyzed 48 h posttransfection. Cell surface CFTR or CFTR mutants were biotinylated using a two-step cell surface periodate/LC-hydrazide biotinylation procedure previously described (5Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (120) Google Scholar). At zero time, both steps were conducted at 4 °C to label the entire surface pool of CFTR. Internalization is monitored by a loss of biotinylation of the cell surface pool by including a 37 °C incubation period (shown on the x axis as time in min) between periodate and biotin LC-hydrazide treatments. Biotinylated and nonbiotinylated proteins were separated on a monomeric avidin column, and CFTR and A1440Xwere in vitro phosphorylated and analyzed by SDS-polyacrylamide gels and autoradiography to quantitate the amount of CFTR remaining on the cell surface during the warm-up step. Each time point represents the mean ± S.E. of 13 experiments for wild-type CFTR and 6 for the A1440X. B, internalization of CFTR and Y1424A in COS-7 cells. Cells transfected with wild-type CFTR (pGT-CFTR) or Y1424A were analyzed as described in A for percentage CFTR internalized from the cell surface during the warm-up period. C, percentage of CFTR or Y1424A at the cell surface under steady-state conditions. Cells transfected with CFTR or Y1424A were analyzed for total CFTR expression by performing the two-step biotinylation reaction without a warm-up step. Biotinylated and nonbiotinylated CFTR or Y1424A was separated on a monovalent avidin column and quantitated as described for A. The percentage of CFTR at the cell surface represents the mean ± S.E. of six experiments. D, wild-type CFTR expression in COS-7 cells using pMT-CFTR and pGT-CFTR. COS-7 cells transfected with CFTR using the pMT-CFTR and pGT-CFTR vector were analyzed 48 h posttransfection. Biotinylated (biotin eluent) and nonbiotinylated (unbound) CFTR was separated on a monovalent avidin column. Biotinylated and nonbiotinylated CFTR were then immunoprecipitated from the two fractions, phosphorylated in vitro with protein kinase A and [γ-32P]ATP, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The positions of bands B and C are indicated on the left. The relative amount of CFTR expressed using the pMT-CFTR vector was always higher than the pGT-CFTR vector.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we tested the only point mutation that affected internalization of the chimeras, Y1424A. Analysis of this mutation in CFTR revealed that it was maturely glycosylated (Fig. 5 B) but internalized 41% more slowly than the wild-type CFTR protein (k e = 0.16 versus 0.27 (p < 0.01); Fig.6 B). Since slower internalization would imply that the steady-state distribution of Y1424A might favor a higher cell surface distribution pattern, we determined the percentage of CFTR at the cell surface using surface biotinylation at 4 °C. As predicted, the percentage of Y1424A at the cell surface was higher than the wild-type CFTR protein (36.1 versus 23.1% (p < 0.01), respectively; Fig. 6 C), supporting the idea
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