FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017283606> ?p ?o ?g. }

• W2017283606 endingPage "23630" @default.
• W2017283606 startingPage "23623" @default.
• W2017283606 abstract "Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides.N-Glycosylation of Asn19 and Asn23provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP. Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides.N-Glycosylation of Asn19 and Asn23provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP. Multidrug resistance is frequently characterized by an ATP-dependent reduction in cellular drug accumulation. This phenotype can occur in mammalian cells by overexpression of either the multidrug resistance protein (MRP) 1The abbreviations used are: MRP, multidrug resistance protein; ABC, ATP-binding cassette; MSD, membrane spanning domain; NBD, nucleotide binding domain; CFTR, cystic fibrosis transmembrane conductance regulator; SUR, sulfonylurea receptor; MOAT, multispecific organic anion transporter; mAb, monoclonal antibody; PCR, polymerase chain reaction; PNGase F, protein N-glycosidase F. or P-glycoprotein (MDR1) (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar, 2Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar, 3Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, 4Loe D.W. Deeley R.G. Cole S.P.C. Eur. J. Cancer. 1996; 32: 945-957Abstract Full Text PDF Scopus (403) Google Scholar, 5Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Biol. 1996; 6: 610-617Google Scholar). MRP and P-glycoprotein belong to the ATP-binding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar). Nevertheless, both proteins confer resistance to a broad range of cytotoxic xenobiotics including doxorubicin, vincristine, and VP-16 (etoposide), drugs that are widely used in the treatment of many human cancers. However, there is growing evidence that the mechanisms by which MRP and P-glycoprotein reduce cellular drug accumulation are not the same, suggesting that there are major differences in the drug-protein interactions of these two molecules (6Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 7Muller M. Meijer C. Zaman G.J.R. Borst P. Scheper R.J. Mulder N.H. de Vries E.G.E. Jansen P.L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar, 8Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar). Like most eukaryotic ABC proteins, MRP and P-glycoprotein contain hydrophobic membrane spanning domains (MSDs) and cytoplasmic nucleotide binding domains (NBDs) (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar). To understand how drugs interact with P-glycoprotein, there has been considerable interest in determining the precise topology of this integral membrane protein. Investigations in most experimental systems support a model in which P-glycoprotein is organized as a symmetrically arranged, tandemly duplicated molecule with each half consisting of six transmembrane segments followed by a NBD (10Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 15414-15419Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), but alternate models have also been proposed (11Zhang J.-T. Ling V. J. Biol. Chem. 1991; 266: 18224-18232Abstract Full Text PDF PubMed Google Scholar, 12Skach W.R. Calayag M.C. Lingappa V.R. J. Biol. Chem. 1993; 268: 6903-6908Abstract Full Text PDF PubMed Google Scholar). At present, little is known about the membrane topology of MRP. The topological model we proposed when MRP was cloned in 1992 was based on computer-assisted hydropathy analyses of its deduced amino acid sequence and alignment with the predicted structure ofltpgpA (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar). LtpgpA is an ABC protein cloned fromLeishmania tarentolae which was the most closely related protein to MRP known at that time (13Ouellette M. Fase-Fowler F. Borst P. EMBO J. 1990; 9: 1027-1033Crossref PubMed Scopus (218) Google Scholar). In the original model, we suggested that MRP consisted of eight transmembrane segments and an NBD in its NH2-proximal half and only four transmembrane segments and an NBD in its COOH-proximal half (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar). More recently, alignment of the hydropathy profiles of human MRP with those of its murine ortholog (14Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar) and several members of the ABC superfamily (including the related sulfonylurea receptor, SUR (15Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. Boyd A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1283) Google Scholar), and the yeast cadmium resistance factor, YCF1 (16Szczypka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar), as well as P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)) suggested to us a different topology for MRP. In this later model, we predicted that MRP contains two MSDs of six transmembrane helices in a “6 + 6” configuration typical of several eukaryotic ABC transporters (9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar), plus an extremely hydrophobic NH2-terminal MSD of approximately 220 amino acids (4Loe D.W. Deeley R.G. Cole S.P.C. Eur. J. Cancer. 1996; 32: 945-957Abstract Full Text PDF Scopus (403) Google Scholar, 14Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 17Cole S.P.C. Deeley R.G. Hait W.N. Drug Resistance. Kluwer Academic Publishers, Norwell, MA1996: 39-62Google Scholar, 18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar). This additional hydrophobic domain is predicted to contain four to six transmembrane segments and is not present in ABC proteins such as P-glycoprotein and CFTR. Thus it is a characteristic feature of members of the MRP branch of the ABC transporter superfamily (4Loe D.W. Deeley R.G. Cole S.P.C. Eur. J. Cancer. 1996; 32: 945-957Abstract Full Text PDF Scopus (403) Google Scholar, 14Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar). One approach to defining the topology of integral membrane glycoproteins is to identify amino acids that are modified byN-linked oligosaccharides (19Chang X.-B. Hou Y.-X. Jensen T.J. Riordan J.R. J. Biol. Chem. 1994; 269: 18572-18575Abstract Full Text PDF PubMed Google Scholar, 20Schinkel A.H. Kemp S. Dolle M. Rudenko G. Wagenaar E. J. Biol. Chem. 1993; 268: 7474-7481Abstract Full Text PDF PubMed Google Scholar). The co-translational attachment of high mannose oligosaccharides to nascent polypeptide chains occurs on asparagine residues in the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline (21Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar, 22Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). This process is carried out by an oligosaccharyltransferase localized on the lumenal side of the endoplasmic reticulum, so that only asparagine residues that are extracytosolic are glycosylated (23Kaplan H.A. Welply J.K. Lennarz W.J. Biochim. Biophys. Acta. 1987; 906: 161-173Crossref PubMed Scopus (136) Google Scholar). We and others (24Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar, 25Krishnamachary N. Center M.S. Cancer Res. 1993; 53: 3658-3661PubMed Google Scholar) have shown that MRP is N-glycosylated with complex-type oligosaccharides. We have also shown recently that limited proteolysis of MRP cleaves the protein in the region connecting the first NBD (NBD1) and the COOH-proximal MSD (MSD3) producing twoN-glycosylated fragments. These observations indicated that asparagine residues in both halves of MRP were modified with oligosaccharides (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar). Bakos et al. (26Bakos E. Hegedus T. Hollo Z. Welker E. Tusnady G.E. Zaman G.J.R. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) have used a similar approach to demonstrate that the NH2-terminal MSD (MSD1) of MRP is glycosylated. However, which specific N-glycosylation sequons in MSD1 and MSD3 are used and whether the middle MSD (MSD2) is also glycosylated is not known. In the present study, we have carried out more extensive analyses of the partial proteolysis products of MRP together with site-directed mutagenesis of selected asparagine residues to identify the sites of N-linked glycosylation in this protein. The results obtained place constraints on the possible topologies of MRP and indicate that the NH2 terminus of the protein is localized to the extracellular surface of the plasma membrane. SV40-transformed Cos-1 cells were obtained from the American Type Culture Collection (Rockville, MD). The MRP-overexpressing, multidrug-resistant H69AR small cell lung cancer cell line has been described previously (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar). T14 cells are a HeLa cell population transfected with an episomally replicating pCEBV7 expression construct containing a full-length cDNA encoding human MRP (3Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). Monoclonal antibody (mAb) QCRL-1 is a murine mAb that reacts with the heptapeptide918SSYSGDI924 in the connector region of MRP (Centocor, Malvern, PA) (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 27Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar). MRP-1 is a rabbit polyclonal antiserum raised against a peptide from NBD1 of MRP (765GVNLSGGQKQRVSLA779) (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar) and was purified as described (24Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar). The rat mAb MRPr1 and the mouse mAb MRPm6 were raised against fusion proteins containing MRP amino acids 194–360 and MRP amino acids 1294–1430 plus 1497–1531, respectively (28Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J.L.M. Scheper R.J. Zaman G.J.R. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar). Both mAbs were kindly provided by Dr. R. J. Scheper (Free University Hospital, Amsterdam, The Netherlands). The locations of membrane spanning helices in human MRP were predicted using the algorithms of Eisenberget al. (29Eisenberg D. Schwarz E. Komaromy M. Wall R. J. Mol. Biol. 1984; 179: 125-142Crossref PubMed Scopus (1708) Google Scholar), Klein et al. (30Klein P. Kanehisa M. DeLisi C. Biochim. Biophys. Acta. 1985; 815: 468-476Crossref PubMed Scopus (628) Google Scholar), Argos et al. (31Argos P. Rao J.K.M. Hargrave P.A. Eur. J. Biochem. 1982; 128: 565-575Crossref PubMed Scopus (210) Google Scholar), and Jones et al. (MEMSAT) (32Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (707) Google Scholar). The topology of MRP was also predicted by the neural network system PredictProtein using a multiple sequence alignment that included human and murine MRP/mrp, human and rabbit multispecific organic anion transporters (MOAT/epithelial basolateral conductance regulator), and yeast YCF1 (33Rost B. Fariselli P. Casadio R. Protein Sci. 1996; 5: 1704-1718Crossref PubMed Scopus (533) Google Scholar). The assembly of a full-length MRP cDNA in pBluescript II KS+ (Stratagene, La Jolla, CA) has been described (2Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar). The full-length cDNA was transferred into pcDNAI/Amp (Invitrogen, San Diego, CA) to generate the pcDNAI-MRP1 expression vector. The asparagine codons of potential N-glycosylation acceptor sites at positions 19, 23, 71, 1006, and 1156 of MRP were converted to glutamine codons by site-directed mutagenesis. The N23Q and N71Q mutations were generated by a modification of the PCR-based megaprimer method (34Boles E. Miosga T. Curr. Genet. 1995; 28: 197-198Crossref PubMed Scopus (23) Google Scholar). A template was prepared by cloning the BamHI fragment from pcDNAI-MRP1 (containing the first 840 nucleotides of MRP coding sequence, plus 86 nucleotides of 5′-untranslated region and a portion of the pcDNAI/Amp multiple cloning site) into pBluescript SK+ in both orientations. The first PCR was carried out on the pBluescript construct containing the BamHI fragment cloned in the EcoRI toHindIII orientation (5′ → 3′) using the M13 primer and either the N23Q (5′-C ACG TGG CAA ACC AGC AAC CCC GAC T-3′) or the N71Q (5′-A CCT CTC CAG AAA ACC AAA ACT GCC T-3′) mutagenic primer (substituted nucleotide positions underlined). The purified PCR product then served as a megaprimer in a second PCR reaction, along with the M13 primer, using the pBluescript construct with the BamHI insert in theHindIII to EcoRI orientation (5′ → 3′) as a template. The final PCR product was then digested with BamHI and cloned back into pBluescript SK+. The N19Q, N1006Q, and N1156Q mutations were generated using the TransformerTM site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) based on the method developed by Deng and Nickoloff (35Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar). The templates for mutagenesis were prepared by cloning the BamHI fragment as above (for N19Q) and the XmaI fragment (MRP nucleotides 2337–4322) (for N1006Q and N1156Q) from pcDNAI-MRP1 into pGEM®-3Z (Promega, Madison, WI). Mutagenesis was then performed according to the manufacturer's instructions using the ScaI/StuI and SspI/EcoRV selection primers (for N19Q and N1006Q/N1156Q mutations, respectively), and the following sense mutagenic oligonucleotide primers: 5′-C TGG GAC TGGCAG GTC ACG TGG-3′ (N19Q), 5′-C CCC ATC GTCCAG GGG ACT CAG G-3′ (N1006Q), and 5′-C TAT TCC CAT TTC CAG GAG ACC TTG C-3′ (N1156Q). The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEM®-3Z as a template. After confirming all mutations by dideoxy sequencing using SequenaseTM version 2.0 (U.S. Biochemical Corp.), DNA fragments containing the desired mutations were transferred back into pcDNAI-MRP1. The N19Q/N23Q/N1006Q triple mutant was prepared by cloning the BamHI fragment containing the N19Q/N23Q mutation into pcDNAI-MRP1/N1006Q. All regions that were synthesized by PCR or by T4 DNA polymerase (when the TransformerTM protocol was used) were confirmed by DNA sequencing of the final constructs. pcDNAI-MRP1 expression vectors encoding wild-type and mutant forms of MRP were transiently transfected into Cos-1 cells using LipofectAMINE (Life Technologies, Inc., Burlington, Ontario, Canada). Briefly, 5 × 105 cells per well were seeded in 6-well plates, and 24 h later, cells were washed and overlaid with 1 ml of serum-free Dulbecco's modified Eagle's medium containing 2 μg of supercoiled DNA and 8 μl of LipofectAMINE. After 4 h, 1 ml of Dulbecco's modified Eagle's medium containing 20% calf serum was added to each well. The medium was replaced after 24 h with 2 ml of fresh Dulbecco's modified Eagle's medium, 10% calf serum, and the cells were incubated for a further 24–48 h before harvesting. Membrane-enriched fractions were prepared as described previously (27Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar). Membrane pellets were resuspended in Tris sucrose buffer (250 mm sucrose, 10 mm Tris, pH 7.5), aliquoted, and frozen at −70 °C. After thawing, membranes were disaggregated by passage through a 27-gauge needle. For proteolysis, membrane proteins were diluted to 2–3 μg/μl in phosphate-buffered saline and incubated at 37 °C with diphenylcarbamyl chloride-treated trypsin (ICN Biomedicals, St. Laurent, Quebec, Canada) at trypsin:membrane protein ratios of 1:30 to 1:810 (w:w) for 30 min. Proteolysis was stopped by the addition of phenylmethylsulfonyl fluoride and leupeptin. After centrifugation, membrane pellets were resuspended in phosphate-buffered saline containing 4.8 mmphenylmethylsulfonyl fluoride. In some experiments, membranes were treated with protein N-glycosidase F (PNGase F) (New England Biolabs, Mississauga, Ontario, Canada) (24Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar). Sodium phosphate, pH 7.5, was added to membrane proteins to a final concentration of 50 mm in the absence of detergents, followed by PNGase F (10–15 New England Biolabs units/μg protein), and the samples were incubated overnight at 37 °C. Samples were solubilized and subjected to SDS-polyacrylamide gel electrophoresis essentially as described (2Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar,24Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar). Immunoblotting was performed as described (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 27Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar), except 4% skim milk powder in TBS-T (10 mm Tris, 0.15 mNaCl, 0.05% Tween 20, pH 7.5) was used as the blocking solution. The amino acid sequence of MRP contains 14N-glycosylation sequons (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar). Of these, eight are in either NBD1 or NBD2 or in the connector region that joins the two halves of the protein (Fig. 1). Previous studies of MRP and other ABC transport proteins indicate that these regions are cytoplasmic (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 19Chang X.-B. Hou Y.-X. Jensen T.J. Riordan J.R. J. Biol. Chem. 1994; 269: 18572-18575Abstract Full Text PDF PubMed Google Scholar, 27Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar, 28Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J.L.M. Scheper R.J. Zaman G.J.R. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar, 36Shapiro A.B. Duthie M. Childs S. Okubo T. Ling V. Int. J. Cancer. 1996; 67: 256-263Crossref PubMed Scopus (12) Google Scholar). Consequently, these eight sites are not expected to be modified with N-linked oligosaccharides. Of the remaining six sequons, three reside in MSD1 (Asn19, Asn23, and Asn71), one is in MSD2 (Asn354), and two are found in MSD3 (Asn1006and Asn1156). Analyses of the MRP sequence using various algorithms for predicting the locations of membrane spanning helices suggested several topological models of MRP (29Eisenberg D. Schwarz E. Komaromy M. Wall R. J. Mol. Biol. 1984; 179: 125-142Crossref PubMed Scopus (1708) Google Scholar, 30Klein P. Kanehisa M. DeLisi C. Biochim. Biophys. Acta. 1985; 815: 468-476Crossref PubMed Scopus (628) Google Scholar, 31Argos P. Rao J.K.M. Hargrave P.A. Eur. J. Biochem. 1982; 128: 565-575Crossref PubMed Scopus (210) Google Scholar). The topology of MSD1 varied according to the algorithm used, with the predicted number of transmembrane segments ranging from four to six (Fig. 1, left, A-D). As a result, differences in the orientation of the NH2 terminus and the potential sites ofN-glycosylation were also predicted. Models A andD possess six and four transmembrane segments, respectively, and the NH2 terminus is cytoplasmic. Consequently, Asn71 is the only potential site ofN-glycosylation. In contrast, MSD1 in model B has five transmembrane segments and an extracytoplasmic NH2terminus. Accordingly, Asn19 and Asn23 are both potential N-glycosylation acceptors and Asn71 is not. Finally, in model C, the positions of the four predicted transmembrane segments place all threeN-glycosylation sequons in the cytosolic NH2terminus, where they are inaccessible for glycosylation. As with MSD1, the predicted topology of MSD2 varied according to the algorithm used. On the basis of previous comparative hydropathy analyses, we and others (4Loe D.W. Deeley R.G. Cole S.P.C. Eur. J. Cancer. 1996; 32: 945-957Abstract Full Text PDF Scopus (403) Google Scholar, 9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar, 14Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 26Bakos E. Hegedus T. Hollo Z. Welker E. Tusnady G.E. Zaman G.J.R. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) have proposed that MSD2 possesses six transmembrane segments, a configuration typical of the comparable domain in the P-glycoproteins and many other ABC proteins (Fig. 1, middle). This topology predicts that Asn354 in MSD2 is localized to the lumenal or extracellular face of the membrane and is thus a potential site ofN-glycosylation. The predictions of the different algorithms for the topology of MSD3 were more consistent than for MSD1 and MSD2. Two of the models suggest that MSD3 contains six transmembrane segments, as we and others have proposed previously (4Loe D.W. Deeley R.G. Cole S.P.C. Eur. J. Cancer. 1996; 32: 945-957Abstract Full Text PDF Scopus (403) Google Scholar, 9Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar, 14Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 26Bakos E. Hegedus T. Hollo Z. Welker E. Tusnady G.E. Zaman G.J.R. Flens M.J. Varadi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) (Fig. 1, right top). In these models, Asn1006 is the only potentialN-glycosylation acceptor in this domain. In contrast, the other two models predict that MSD3 possesses only four transmembrane segments, as proposed in our original “8 + 4” model of MRP (1Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3010) Google Scholar) (Fig. 1, right bottom). According to these models, both Asn1006 and Asn1156 are extracytosolic and thus potential sites for N-glycosylation. To localize the approximate sites ofN-glycosylation, MRP-enriched membranes were digested with trypsin and immunoblotted with antibodies known to react with specific regions of MRP. These included the MRP-1 antiserum raised against the synthetic peptide 765GVNLSGGQKQRVSLA779 in NBD1 (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar); mAb QCRL-1 which reacts with the human MRP-specific heptapeptide918SSYSGDI924 in the cytoplasmic connector region (18Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P.C. Cancer Res. 1996; 56: 3307-3314PubMed Google Scholar, 27Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar); mAb MRPr1 (28Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J.L.M. Scheper R.J. Zaman G.J.R. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar) whose epitope lies between amino acids 229–281 2R. G. Deeley and S. P. C. Cole, unpublished results ; and mAb MRPm6 (28Flens M.J. Izquierdo M.A. Scheffer G.L. Fritz J.M. Meijer C.J.L.M. Scheper R.J. Zaman G.J.R. Cancer Res. 1994; 54: 4557-4563PubMed Google Scholar) which reacts with a cytoplasmic COOH-terminal formic acid fragment of MRP (amino acids 1389–1531).2 In preliminary experiments, we determined that no proteolytic fragments of MRP were observed after incubation of MRP-transfected HeLa cells with trypsin unless the cells were first permeabilized with digitonin. Since MRP has been shown to be targeted to the plasma membrane in these cells (24Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar), this indicated that all sites of trypsin digestion we have detected are intracellular (not shown). In subsequent experiments, tryptic digests were carried out on crude membrane preparations. MRP-enriched crude membranes from H69AR cells were digested with trypsin at increasing trypsin to membrane protein ratios up to 1:30 (w:w), and the proteolytic products were detected by immunoblotting (Fig. 2). Initially, two polypeptides are produced by digestion of a portion of the connector region of MRP. One is a fragment of approximately 120-kDa detected by mAb MRPr1 and MRP-1 antiserum (and to a lesser extent by mAb QCRL-1) which corresponds to the NH2-proximal half of MRP (i.e. MSD1 + MSD2 + NBD1) (labeled N-1) (Fig. 2, A–C). The second is a fragment of 75–80 kDa detected by mAbs QCRL-1 and MRPm6 corresponding to the COOH-proximal half of MRP (i.e. MSD3 + NBD2) (labeled C-1) (Fig. 2, C and D). We have shown previously that the epitope for mAb QCRL-1 (amino acids 918–924) li" @default.
• W2017283606 created "2016-06-24" @default.
• W2017283606 creator A5009648988 @default.
• W2017283606 creator A5039554395 @default.
• W2017283606 creator A5039918356 @default.
• W2017283606 creator A5044399566 @default.
• W2017283606 creator A5057120814 @default.
• W2017283606 creator A5066932251 @default.
• W2017283606 creator A5089670266 @default.
• W2017283606 date "1997-09-01" @default.
• W2017283606 modified "2023-10-14" @default.
• W2017283606 title "Membrane Topology of the Multidrug Resistance Protein (MRP)" @default.
• W2017283606 cites W1526754730 @default.
• W2017283606 cites W1528658828 @default.
• W2017283606 cites W1530873901 @default.
• W2017283606 cites W1561829393 @default.
• W2017283606 cites W1571379445 @default.
• W2017283606 cites W1603254979 @default.
• W2017283606 cites W1647833960 @default.
• W2017283606 cites W1678488130 @default.
• W2017283606 cites W171632912 @default.
• W2017283606 cites W1716367672 @default.
• W2017283606 cites W1890023926 @default.
• W2017283606 cites W1975502332 @default.
• W2017283606 cites W1977254345 @default.
• W2017283606 cites W1986381180 @default.
• W2017283606 cites W1995263127 @default.
• W2017283606 cites W2004804291 @default.
• W2017283606 cites W2006677268 @default.
• W2017283606 cites W2019710821 @default.
• W2017283606 cites W2024988940 @default.
• W2017283606 cites W2026582002 @default.
• W2017283606 cites W2029335288 @default.
• W2017283606 cites W2040198458 @default.
• W2017283606 cites W2048034139 @default.
• W2017283606 cites W2051864088 @default.
• W2017283606 cites W2057589234 @default.
• W2017283606 cites W2069274485 @default.
• W2017283606 cites W2074886854 @default.
• W2017283606 cites W2083669966 @default.
• W2017283606 cites W2087344385 @default.
• W2017283606 cites W2089606003 @default.
• W2017283606 cites W2125558238 @default.
• W2017283606 cites W2148959338 @default.
• W2017283606 cites W2152708819 @default.
• W2017283606 cites W2154283357 @default.
• W2017283606 cites W2162097287 @default.
• W2017283606 cites W2168070336 @default.
• W2017283606 cites W2168095256 @default.
• W2017283606 doi "https://doi.org/10.1074/jbc.272.38.23623" @default.
• W2017283606 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9295302" @default.
• W2017283606 hasPublicationYear "1997" @default.
• W2017283606 type Work @default.
• W2017283606 sameAs 2017283606 @default.
• W2017283606 citedByCount "211" @default.
• W2017283606 countsByYear W20172836062012 @default.
• W2017283606 countsByYear W20172836062013 @default.
• W2017283606 countsByYear W20172836062014 @default.
• W2017283606 countsByYear W20172836062015 @default.
• W2017283606 countsByYear W20172836062016 @default.
• W2017283606 countsByYear W20172836062017 @default.
• W2017283606 countsByYear W20172836062018 @default.
• W2017283606 countsByYear W20172836062019 @default.
• W2017283606 countsByYear W20172836062020 @default.
• W2017283606 countsByYear W20172836062021 @default.
• W2017283606 countsByYear W20172836062022 @default.
• W2017283606 crossrefType "journal-article" @default.
• W2017283606 hasAuthorship W2017283606A5009648988 @default.
• W2017283606 hasAuthorship W2017283606A5039554395 @default.
• W2017283606 hasAuthorship W2017283606A5039918356 @default.
• W2017283606 hasAuthorship W2017283606A5044399566 @default.
• W2017283606 hasAuthorship W2017283606A5057120814 @default.
• W2017283606 hasAuthorship W2017283606A5066932251 @default.
• W2017283606 hasAuthorship W2017283606A5089670266 @default.
• W2017283606 hasBestOaLocation W20172836061 @default.
• W2017283606 hasConcept C114614502 @default.
• W2017283606 hasConcept C114851261 @default.
• W2017283606 hasConcept C12554922 @default.
• W2017283606 hasConcept C133936738 @default.
• W2017283606 hasConcept C144647389 @default.
• W2017283606 hasConcept C184720557 @default.
• W2017283606 hasConcept C185592680 @default.
• W2017283606 hasConcept C33923547 @default.
• W2017283606 hasConcept C41008148 @default.
• W2017283606 hasConcept C41625074 @default.
• W2017283606 hasConcept C54355233 @default.
• W2017283606 hasConcept C55493867 @default.
• W2017283606 hasConcept C86803240 @default.
• W2017283606 hasConcept C95444343 @default.
• W2017283606 hasConceptScore W2017283606C114614502 @default.
• W2017283606 hasConceptScore W2017283606C114851261 @default.
• W2017283606 hasConceptScore W2017283606C12554922 @default.
• W2017283606 hasConceptScore W2017283606C133936738 @default.
• W2017283606 hasConceptScore W2017283606C144647389 @default.
• W2017283606 hasConceptScore W2017283606C184720557 @default.
• W2017283606 hasConceptScore W2017283606C185592680 @default.
• W2017283606 hasConceptScore W2017283606C33923547 @default.
• W2017283606 hasConceptScore W2017283606C41008148 @default.

• next