The Transmembrane Domains of the Human Multidrug Resistance P-glycoprotein Are Sufficient to Mediate Drug Binding and Trafficking to the Cell Surface
1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês
10.1074/jbc.274.35.24759
ISSN1083-351X
Autores Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoThe human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1–379 plus 681–1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1–379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface. The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1–379 plus 681–1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1–379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface. P-glycoprotein P-gp residues 1–682, containing transmembrane segments 1–6 and the first nucleotide-binding domain P-gp residues 681–1280, containing transmembrane segments 7–12 and the second nucleotide-binding domain residues 1–1023 of P-gp residues 1–379 and 681–1025 of P-gp P-gp residues 1–379, containing transmembrane segments 1–6 P-gp residues 681–1025, containing transmembrane segments 7–12 transmembrane l-1-tosylamido-2-phenylethylchloromethyl ketone tetraphenylarsonium Epstein-Barr virus nuclear antigen 1 isopropyl-β-d-thiogalactopyranoside The human multidrug resistance P-glycoprotein (P-gp)1 is an ATP-dependent drug pump that extrudes a broad range of hydrophobic substrates from the cell (reviewed in Refs. 1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar and 2Germann U.A. Chambers T.C. Cytotechnology. 1998; 27: 31-60Crossref PubMed Google Scholar). Its likely physiological role is to protect the vital organs of the body from the cytotoxic effects of exogenous and endogenous compounds (3Schinkel A.H. Smit J.J. van Tellingen O. Beijnen J.H. Wagenaar E. van Deemter L. Mol C.A. van der Valk M.A. Robanus-Maandag E.C. te Riele H.P.J. Berns A.J.M. Borst P. Cell. 1994; 77: 491-502Abstract Full Text PDF PubMed Scopus (2089) Google Scholar, 4Sparreboom A. van Asperen J. Mayer U. Schinkel A.H. Smit J.W. Meijer D.K. Borst P. Nooijen W.J. Beijnen J.H. van Tellingen O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2031-2035Crossref PubMed Scopus (857) Google Scholar, 5Charuk J.H. Grey A.A. Reithmeier R.A. Am. J. Physiol. 1998; 274: F1127-F1139PubMed Google Scholar). The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during cancer (6Chan H.S. Grogan T.M. DeBoer G. Haddad G. Gallie B.L. Ling V. Eur. J. Cancer. 1996; 6: 1051-1061Abstract Full Text PDF Scopus (29) Google Scholar, 7Fisher G.A. Lum B.L. Hausdorff J. Sikic B.I. Eur. J. Cancer. 1996; 6: 1082-1088Abstract Full Text PDF Scopus (134) Google Scholar) and AIDS chemotherapy (8Lee C.G. Gottesman M.M. Cardarelli C.O. Ramachandra M. Jeang K.T. Ambudkar S.V. Pastan I. Dey S. Biochemistry. 1998; 37: 3594-3601Crossref PubMed Scopus (460) Google Scholar, 9Kim R.B. Fromm M.F. Wandel C. Leake B. Wood A.J. Roden D.M. Wilkinson G.R. J. Clin. Invest. 1998; 101: 289-294Crossref PubMed Scopus (1040) Google Scholar). P-gp, encoded by the MDR1 gene, has 1280 amino acids organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids (10Chen C.J. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1721) Google Scholar). Each repeat has an N-terminal hydrophobic domain containing six transmembrane sequences followed by a hydrophilic domain containing a nucleotide binding site (11Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 12Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The protein is a member of the ABC (ATP-binding cassette) superfamily of transporters (13Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar). There has been considerable effort in understanding the role of various domains in the mechanism of transport by P-gp. Both halves of P-gp have been expressed as separate polypeptides, but substrate-stimulated ATPase activity was detected only when the two halves were expressed simultaneously (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar). The nucleotide-binding domains have been expressed in bacteria, and can bind ATP and its analogues (15Shimabuku A.M. Nishimoto T. Ueda K. Komano T. J. Biol. Chem. 1992; 267: 4308-4311Abstract Full Text PDF PubMed Google Scholar, 16Baubichon-Cortay H. Baggetto L.G. Dayan G. Di Pietro A. J. Biol. Chem. 1994; 269: 22983-22989Abstract Full Text PDF PubMed Google Scholar, 17Sharma S. Rose D.R. J. Biol. Chem. 1995; 270: 14085-14093Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 18Dayan G. Baubichon-Cortay H. Jault J.M. Cortay J.C. Deleage G. Di Pietro A. J. Biol. Chem. 1996; 271: 11652-11658Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Both ATP-binding sites are essential because inactivation of either site by mutagenesis or chemical modification inhibits substrate-stimulated ATPase activity (19Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 20Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. 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Chem. 1995; 270: 5441-5448Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), cysteine-scanning mutagenesis studies using a thiol-reactive substrate (27Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), and mutational analysis studies (28Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (190) Google Scholar, 29Kajiji S. Talbot F. Grizzuti K. Van Dyke-Phillips V. Agresti M. Safa A.R. Gros P. Biochemistry. 1993; 32: 4185-4194Crossref PubMed Scopus (90) Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 31Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (127) Google Scholar) suggest that TM6 and TM12 are particularly important for drug-protein interactions. These two segments are close to each other in the tertiary structure of P-gp (32Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 33Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). There is evidence, however, that suggests that the nucleotide binding domains also contribute to drug binding. For example, mutations in the nucleotide-binding domains of mouse mdr3 P-gp have also been reported to cause large alterations in the drug resistance phenotypes in transfected cells (34Beaudet L. Gros P. J. Biol. Chem. 1995; 270: 17159-17170Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Recently, it was reported that the nucleotide-binding domains could also interact with hydrophobic molecules such as steroids and flavonoids (35Conseil G. Baubichon-Cortay H. Dayan G. Jault J.M. Barron D. Di Pietro A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9831-9836Crossref PubMed Scopus (376) Google Scholar). The aim of our study was to determine if P-gp deletion mutants missing one or both nucleotide-binding domains could still interact with drug substrates. An in vivo assay was used to test for drug binding by the deletion mutants. The rationale for this approach was that if the transmembrane domains alone could form the drug-binding site, then expression in the presence of drug substrates should result in a tightly and correctly folded protein that would be trafficked to the cell surface. The cDNAs coding for full-length MDR1 (36Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar), mutant P709G (37Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 28683-28689Abstract Full Text PDF PubMed Google Scholar), or the N-half and C-half P-gp molecules (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and containing the epitope for monoclonal antibody A52 at the C-terminal ends were subcloned into the mammalian expression vector pMT21. The cDNAs coding for TMD1 (residues 1–379) and TMD2 (residues 681–1025) tagged at the C-end with A52 (38Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) were joined together to give TMD1+2 (residues 1–379 and 681–1025; transmembrane segments 1–6 and 7–12 only) and inserted into pMT21. P-gp cDNA coding for residues 1–1023 (i.e. deletion of the second nucleotide-binding domain) and tagged with A52 at the C-end was inserted into vector pMT21 to give the ΔNBF2 construct. For expression studies involving drug resistance assays, the cDNAs of the A52-tagged wild-type, mutant P709G, N-half, C-half, and TMD1+2 were also inserted into the Epstein-Barr virus-based vector, pREP4 (Invitrogen Inc.). For simple expression studies, HEK 293 cells were transfected with the cDNAs coding for the mutant P-gps. After 48 h, the cells were lysed with SDS sample buffer (63 mm Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 2% (v/v) β-mercaptoethanol) containing 50 mm EDTA and protease inhibitors (10 μm E-64, 12 μg/ml leupeptin, 100 units/ml aprotinin, 50 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 25 μg/ml benzamidine). The samples were then subjected to SDS-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and developed with monoclonal antibody A52 (36Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar) or a rabbit anti-P-gp polyclonal antibody that is specific for the N-terminal nucleotide-binding domain (38Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), followed by enhanced chemiluminescence (Life Technologies, Inc.). For drug resistance assays, HEK 293-EBNA cells (Invitrogen) constitutively expressing the Epstein-Barr virus nuclear antigen 1 (EBNA-1) gene and resistant to neomycin (G418) were used. The HEK 293(EBNA-1) cells were transfected with the mutant cDNA constructs in vector pREP4 (Invitrogen). After 24 h, the medium was replaced with fresh medium containing 10 μm cyclosporin A. Forty-eight hours after transfection, the medium was again replaced with fresh medium containing various concentrations of vinblastine. Then 72 h after transfection, the medium was again replaced with fresh medium containing no drug substrates. After another 4 days, the concentration of vinblastine that caused inhibition of cell growth by 50% was determined as described previously (36Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar). HEK 293(EBNA-1) cells were transfected with the cDNA coding for mutant TMD1+2 in pREP4 and grown for 24 h in the presence or absence of 10 μmcyclosporin A as described above. Membranes were prepared from the transfected cells (22Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) and suspended in TBS (10 mmTris-HCl, pH 7.4, 150 mm NaCl). The membranes (5 mg/ml protein) were treated for 5 min at 22 °C with various amounts of TPCK-treated trypsin (Sigma; 12,000 BAEE units/mg), and the reaction was stopped by the addition of lima bean trypsin inhibitor (Worthington). Digestion with endoglycosidase Hf (New England Biolabs) or with endoglycosidase F (PNGase F; New England Biolabs) was carried out as described previously (39Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 15414-15419Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). HEK 293 cells were transfected with mutant TMD1+2 cDNA and then treated for 24 h with or without 10 μm cyclosporin A or 25 μm verapamil. The cells were chilled, washed twice with ice-cold PBS (phosphate-buffered saline, pH 7.4), and then incubated in the dark with phosphate-buffered saline containing 10 mm NaIO4 for 10 min. The cells were washed with phosphate-buffered saline and then treated for 10 min with 2 mm biotin-LC-hydrazide (Pierce) in 100 mm sodium acetate buffer, pH 5.5. The cells were washed once with 100 mm sodium acetate buffer, pH 5.5, and then solubilized with Tris-buffered saline, pH 7.4 containing 1% (w/v) Triton X-100. Biotinylated TMD1+2 was immunoprecipitated with monoclonal antibody A52. The immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred onto a sheet of nitrocellulose, and probed with streptavidin-conjugated horseradish peroxidase. The cDNAs coding for the A52-tagged TMD1+2 P-gp mutant or the A52-tagged TMD1+2 in which residues 1–27 were deleted were inserted into the Nhe I to Xho I sites of vector pET-21a(+) (Novagen Inc.), transformed into E. coli BL21(DE3) (Novagen), and selected in the presence of 100 μg/ml ampicillin. The clones were grown in LB medium containing 100 μg/ml ampicillin and then grown for 2 h at 37 °C in the presence of various concentrations (0–1 mm) of isopropyl-β-d-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation at 12,000 ×g for 5 min and then lysed with SDS sample buffer as described above. Equivalent volumes of samples were subjected to Western blot analysis, and the mutant P-gps were detected with monoclonal antibody A52 followed by enhanced chemiluminescence. Resistance to tetraphenylarsonium chloride (TPA+) (Sigma) was done as described by Bibi et al. (40Bibi E. Gros P. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9209-9213Crossref PubMed Scopus (58) Google Scholar). Briefly, the same number of E. coli BL21(DE3) cells expressing the mutant TMD1+2 or TMD1+2 missing residues 1–27 were plated on LB agar plates containing 100 μg/ml ampicillin, 100 μm IPTG, and various concentrations (0–5 mm) of TPA+. The number of colonies in each plate was determined after several (1Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar, 2Germann U.A. Chambers T.C. Cytotechnology. 1998; 27: 31-60Crossref PubMed Google Scholar, 3Schinkel A.H. Smit J.J. van Tellingen O. Beijnen J.H. Wagenaar E. van Deemter L. Mol C.A. van der Valk M.A. Robanus-Maandag E.C. te Riele H.P.J. Berns A.J.M. Borst P. Cell. 1994; 77: 491-502Abstract Full Text PDF PubMed Scopus (2089) Google Scholar, 4Sparreboom A. van Asperen J. Mayer U. Schinkel A.H. Smit J.W. Meijer D.K. Borst P. Nooijen W.J. Beijnen J.H. van Tellingen O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2031-2035Crossref PubMed Scopus (857) Google Scholar, 5Charuk J.H. Grey A.A. Reithmeier R.A. Am. J. Physiol. 1998; 274: F1127-F1139PubMed Google Scholar, 6Chan H.S. Grogan T.M. DeBoer G. Haddad G. Gallie B.L. Ling V. Eur. J. Cancer. 1996; 6: 1051-1061Abstract Full Text PDF Scopus (29) Google Scholar, 7Fisher G.A. Lum B.L. Hausdorff J. Sikic B.I. Eur. J. Cancer. 1996; 6: 1082-1088Abstract Full Text PDF Scopus (134) Google Scholar) days at 30 °C and compared with the plates with the control cells (vector only). For measuring resistance to TPA+ in liquid culture, the same number of E. coli BL21(DE3) cells expressing the mutant TMD1+2 or TMD1+2 missing residues 1–27 were grown at 37 °C in LB medium containing 100 μg/ml ampicillin, 100 μm IPTG, and various concentrations (0–5 mm) of TPA+. At hourly intervals, the absorbance at 600 nm was measured and compared with cells containing only the vector. Each of the nucleotide-binding domains of P-gp (Fig.1 A) is predicted to contain 275–300 residues. To examine the role of the various domains of P-gp in drug-protein interactions, we first constructed a mutant that lacked the C-terminal nucleotide-binding domain (ΔNBF2; Fig.1 B). The ΔNBF2 mutant consisted of residues 1–1023 followed by the A52 antibody epitope tag. The presence of the tag facilitated detection of the protein after expression in HEK 293 cells and allowed us to distinguish it from any endogenous expression of P-gp. As shown in Fig. 2 A (lane 1), immunoblot analysis of whole cell extracts of HEK 293 cells transfected with ΔNBF2 mutant cDNA showed the presence of a protein of 110 kDa as the major product. The 110-kDa ΔNBF2 protein was sensitive to digestion by endoglycosidase H (data not shown), suggesting that it was present mainly as a core-glycosylated immature protein.Figure 2Effect of drug substrates on expression of P-gp deletion mutants. HEK 293 cells transfected with the cDNAs of mutant ΔNBF2 (A) or mutant TMD1+2 (B) were treated for 24 h with (+) or without (−) 3 μm cyclosporin A (Cyclo), 25 μmverapamil (Ver), 6 μm vinblastine (Vin), or 100 μm capsaicin (Caps). The cells were then solubilized with SDS sample buffer and subjected to immunoblot analysis with monoclonal antibody A52 followed by enhanced chemiluminescence. The locations of the 130- and 110-kDa (A) and the 100- and 80-kDa (B) proteins are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether the ΔNBF2 mutant protein retained the ability to interact with drug substrates, we used an in vivo drug-binding assay. The rationale was that the ΔNBF2 mutant protein was not completely folded, since it was still sensitive to digestion by endoglycosidase H, and it most likely failed to be transported to the plasma membrane. We had previously shown that some point mutations in P-gp can cause malfolding of the protein such that it remains as a core-glycosylated protein that is not present at the cell surface. By expressing these mutant proteins in the presence of drug substrates, it was possible to induce the mutant proteins to fold in an active conformation, become resistant to endoglycosidase H, and be transported to the plasma membrane (41Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Accordingly, we expressed the 110-kDa ΔNBF2 protein in the presence of various substrates. Fig.2 B shows that expression in the presence of 3 μm cyclosporin A (lane 2), 25 μm verapamil (lane 4), 6 μm vinblastine (lane 6), or 100 μm capsaicin (lane 8) all resulted in the appearance of another polypeptide of apparent mass 130 kDa. The 130-kDa ΔNBF2 P-gp protein was resistant to digestion by endoglycosidase H but not to endoglycosidase F (data not shown). The appearance of the 130-kDa protein in the presence of drug substrates suggests that the 110-kDa ΔNBF2 protein retained the ability to bind drug substrates, resulting in maturation of the protein. We then tested whether a deletion mutant that was missing both nucleotide-binding domains, and containing only the transmembrane segments 1–6 and 7–12 (TMD1+2; Fig. 1 C) could also interact with drug substrates. The cDNA for mutant TMD1+2 was expressed in HEK 293 cells in the presence or absence of drug substrates. In the absence of drug substrates, the major product for TMD1+2 was an 80-kDa protein (Fig. 2 B, lanes 1, 3, 5, and 7). Expression of mutant TMD1+2 in the presence of 3 μm cyclosporin A (lane 2); 25 μm verapamil (lane 4), 6 μm vinblastine (lane 6), or 100 μm capsaicin (lane 8), however, resulted in the appearance of two products of 80 and 100 kDa. These results suggest that the mutant TMD1+2 protein could interact with drug substrates and undergo maturation. The mutant TMD1+2 protein was treated with endoglycosidases to test whether expression in the presence of drug substrates had indeed induced maturation of the protein. Fig. 3 shows that the 80-kDa TMD1+2 protein was sensitive to digestion by endoglycosidase H when expressed in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of drug substrate (cyclosporin A). The 80-kDa protein decreased in apparent mass to 72 kDa following digestion by endoglycosidase H. Similar results were obtained when the 80-kDa protein was digested with endoglycosidase F (Fig. 3, lanes 5–8). These results suggest that the 80-kDa protein is the core-glycosylated immature form. In contrast, synthesis in the presence of drug substrate resulted in the appearance of the 100-kDa protein that was insensitive to digestion by endoglycosidase H (Fig. 3, lanes 1 and 2) but not to endoglycosidase F (Fig. 3, lanes 5 and 6). Apparently, the expression of mutant TMD1+2 in the presence of substrate allowed the mutant protein to leave the endoplasmic reticulum and pass through the Golgi apparatus for extensive modification of the carbohydrate groups. Cells expressing TMD1+2 were then subjected to cell surface labeling to determine if the mutant protein was correctly targeted to the cell surface. Cells expressing the A52-tagged TMD1+2 protein were grown for 24 h in the presence or absence of cyclosporin A, treated with sodium periodate to convert the carbohydrate moieties to aldehydes, and then reacted with biotin-LC-hydrazide (Pierce). Biotin-LC-hydrazide is a cell-impermeant compound that covalently attaches biotin groups to glycoproteins after periodate oxidation of the carbohydrate moieties. The biotinylated TMD1+2 proteins were immunoprecipitated with monoclonal antibody A52 and subjected to Western blot analysis with streptavidin-conjugated horseradish peroxidase, followed by enhanced chemiluminescence. Fig. 4 shows that the mutant TMD1+2 protein (100 kDa) was detected at the cell surface after expression in the presence of cyclosporin A (lane 2) or verapamil (lane 4). There was no labeling of TMD1+2 (80 kDa) when expressed in the absence or presence of drug substrate (lanes 1 and 3). These results showed that TMD1+2 is capable of interacting with drug substrates, resulting in maturation of the carbohydrate residues and trafficking to the cell surface. These results also suggested that the presence of drug substrates during synthesis induces structural changes in the protein. We previously showed that immature and mature forms of full-length wild-type P-gp were different in their sensitivity to digestion by trypsin (42Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Core-glycosylated P-gp was about 100-fold more sensitive to digestion by trypsin compared with the mature enzyme. This enhanced sensitivity to trypsin of the core-glycosylated form of P-gp suggested that it existed in a more "relaxed" conformation than the mature enzyme. Accordingly, the TMD1+2 protein was subjected to trypsin digestion to test for potential structural differences between the 80- and 100-kDa forms of the protein. Membranes prepared from cells expressing the A-52-tagged TMD1+2 protein were treated with trypsin and then subjected to Western blot analysis with monoclonal antibody A52. Fig. 5 (lane 1) shows that the 80-kDa form of TMD1+2 is the major product in the membranes of cells grown in the absence of drug substrate, while the 100-kDa form is the major product when grown in the presence of substrate. In addition, the 80-kDa form of the protein showed relatively higher sensitivity to trypsin (10 μg/ml trypsin;lane 3) than the 100-kDa form of the protein (1000 μg/ml trypsin; lane 5). These results suggested that the trypsin-sensitive sites are more readily accessible in the 80-kDa protein but are probably hidden in the 100-kDa protein as a result of tighter or proper folding when synthesized in the presence of drug substrate. We then tested whether TMD1+2 could confer resistance to cytotoxic drugs when expressed in mammalian cells. The usual approach for determining if a mutant P-gp can confer resistance to different drugs is to generate stable cell lines. The cells are first transfected with the mutant P-gp cDNA followed by selection of drug-resistant colonies after incubation in different cytotoxic drug substrates. A technical problem in using this method for studying mutant TMD1+2 is that the protein does not reach the cell surface in the absence of substrate. Compounding this problem is the observation that the concentrations of drug substrate required for trafficking of TMD1+2 to the cell surface would initially cause extensive cell death, thus complicating the results. Another problem with generating stable lines is that other mechanisms of drug resistance (endogenous) could develop during the relatively long selection period (months) needed to generate highly drug-resistant colonies. To circumvent these potential problems, we developed a faster assay for measuring P-gp-mediated drug resistance that does not require the generation of drug-resistant stable cell lines. The assay relies on the use of an Epstein-Barr virus vector, pREP4, that can be maintained extrachromosomally in HEK 293 cells that constitutively express EBNA-1. The cDNAs for the A52-tagged wild-type, and P-gp mutants P709G, N-half, C-half, and TMD1+2 were inserted into the pREP4 vector and transfected into HEK 293(EBNA-1) cells. Mutant P709G was included because it shows similar processing defects as mutant TMD1+2. When mutant P709G is expressed in HEK 293 cells in the absence of drug substrate, the major product is a protein of 150 kDa that is retained in the endoplasmic reticulum as a core-glycosylated intermediate (37Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 28683-28689Abstract Full Text PDF PubMed Google Scholar). Expression in the presence of drug substrate, however, corrects this processing defect (Fig. 6 A). The P-gp half-molecules were also included because the processing of the N-half is also sensitive to the presence of substrate. When N-half P-gp is expressed alone or co-expressed with the C-half P-gp in the absence of drug substrate, it is retained in the endoplasmic reticulum as a core-glycosylated intermediate. Maturation of the N-half P-gp and trafficking to the cell surface is restored when it is expressed in the presence of the C-half P-gp and drug substrate (42Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Fig. 6 A shows expression of the P-gp mutants using the pREP4 vector and HEK 293(EBNA-1) cells. The major product for wild-type P-gp was the 170-kDa mature protein in the presence or absence of cyclosporin A. For mutant P709G, the major product in the absence of drug substrate was the 150-kDa (core-glycosylated) protein, while the 170-kDa protein was the major product in the presence of substrate. When N-half (90-kDa) P-gp was co-expressed with C-half P-gp in the presence
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