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• W218221756 abstract "P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp. P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp. The ATP-binding cassette (ABC) 2The abbreviations used are: ABCATP-binding cassetteP-gpP-glycoproteinNBDnucleotide-binding domainHEKhuman embryonic kidneyTMtransmembraneTMDtransmembrane domainICLintracellular loopIHintracellular helix. family is the largest class of transmembrane (TM) proteins (1Rees D.C. Johnson E. Lewinson O. ABC transporters: the power to change.Nat. Rev. Mol. Cell Biol. 2009; 10: 218-227Crossref PubMed Scopus (888) Google Scholar). They use ATP hydrolysis to translocate a wide variety of substrates including metabolic products, lipids, peptides, sterols, ions, and drugs across extra- and intracellular membranes. ATP-binding cassette P-glycoprotein nucleotide-binding domain human embryonic kidney transmembrane transmembrane domain intracellular loop intracellular helix. Human ABC proteins have been the subjects of intense research efforts since most are clinically important. Cystic fibrosis (CF), gout, intrahepatic cholestasis types 2 and 3 (liver bile diseases), Tangier disease (HDL deficiency), Dubin-Johnson syndrome (jaundice), hyperinsulimic hypoglycemia of infancy, pseudoxanthoma elasticum, multidrug resistance, secretory diarrheas, anemia, diabetes, and atherosclerosis are examples of potentially lethal or debilitating conditions caused by genetic mutations or altered activity of one of the 48 human ABC proteins (2Borst P. Elferink R.O. Mammalian abc transporters in health and disease.Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1346) Google Scholar). The most common genetic defect is expression of an ABC processing mutant that is defective in folding and trafficking. The classic example is the ΔF508-CFTR (ABCC7) mutant in CF (3Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5935) Google Scholar). Other examples includes ABCG2 (gout), ABCB4/ABCB11 (progressive familial intrahepatic cholestasis), ABCA1 (Tangier disease), ABCC2 (Dubin-Johnson syndrome), ABCC6 (pseudoxanthoma elasticum), and ABCC8 (hyperinsulimic hypoglycemia of infancy) (4Nakagawa H. Toyoda Y. Wakabayashi-Nakao K. Tamaki H. Osumi M. Ishikawa T. Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.J. Pharm. Sci. 2011; 100: 3602-3619Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A high priority is to understand how processing mutations impact synthesis of ABC proteins and use this knowledge to develop therapies to repair the defects. Human ABC proteins appear to be particularly sensitive to point mutations because they are multi-domain membrane proteins (core structure of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs)) that require formation of specific domain-domain contacts to adopt a native structure (5Ostedgaard L.S. Rich D.P. DeBerg L.G. Welsh M.J. Association of domains within the cystic fibrosis transmembrane conductance regulator.Biochemistry. 1997; 36: 1287-1294Crossref PubMed Scopus (59) Google Scholar). ABC proteins differ from other multi-domain proteins because much of the folding occurs post-translationally (6Du K. Sharma M. Lukacs G.L. The ΔF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR.Nat. Struct. Mol. Biol. 2005; 12: 17-25Crossref PubMed Scopus (281) Google Scholar, 7Loo T.W. Bartlett M.C. Clarke D.M. Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).J. Biol. Chem. 2008; 283: 28190-28197Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar8Okiyoneda T. Lukacs G.L. Fixing cystic fibrosis by correcting CFTR domain assembly.J. Cell Biol. 2012; 199: 199-204Crossref PubMed Scopus (36) Google Scholar). The human P-glycoprotein (P-gp) drug pump has been a very useful model system for studying repair of ABC protein defective in processing/folding because processing mutations throughout the molecule can be repaired by expression in the presence of drug substrates (9Loo T.W. Clarke D.M. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) or by introduction of arginine suppressor mutations into TM segments (10Loo T.W. Bartlett M.C. Clarke D.M. Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.J. Biol. Chem. 2007; 282: 32043-32052Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 11Loo T.W. Bartlett M.C. Clarke D.M. Identification of residues in the drug-translocation pathway of the human multidrug resistance P-glycoprotein by arginine mutagenesis.J. Biol. Chem. 2009; 284: 24074-24087Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). P-gp is a classic ABC protein as it is a single polypeptide of 1280 amino acids that forms a structure containing two NBDs and two TMDs. Its physiological role is block entry or export toxic compounds out of the body. The interfaces between the domains play critical roles in the P-gp drug efflux mechanism. Drug substrates bind within a cavity located at the interface between the TMDs (11Loo T.W. Bartlett M.C. Clarke D.M. Identification of residues in the drug-translocation pathway of the human multidrug resistance P-glycoprotein by arginine mutagenesis.J. Biol. Chem. 2009; 284: 24074-24087Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar12Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10594-10599Crossref PubMed Scopus (356) Google Scholar, 13Lugo M.R. Sharom F.J. Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs.Biochemistry. 2005; 44: 14020-14029Crossref PubMed Scopus (78) Google Scholar, 14Lugo M.R. Sharom F.J. Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site.Biochemistry. 2005; 44: 643-655Crossref PubMed Scopus (96) Google Scholar15Loo T.W. Bartlett M.C. Clarke D.M. Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein.J. Biol. Chem. 2003; 278: 39706-39710Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Two ATP-binding sites are located at the interface between the NBDs. ATP hydrolysis occurs by an alternating site mechanism (16Loo T.W. Clarke D.M. Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug- stimulated ATPase activity.J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar17Sauna Z.E. Kim I.W. Nandigama K. Kopp S. Chiba P. Ambudkar S.V. Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-γS, a nonhydrolyzable analogue of ATP.Biochemistry. 2007; 46: 13787-13799Crossref PubMed Scopus (86) Google Scholar, 18Siarheyeva A. Liu R. Sharom F.J. Characterization of an asymmetric occluded state of P-glycoprotein with two bound nucleotides: implications for catalysis.J. Biol. Chem. 2010; 285: 7575-7586Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Delannoy S. Urbatsch I.L. Tombline G. Senior A.E. Vogel P.D. Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.Biochemistry. 2005; 44: 14010-14019Crossref PubMed Scopus (42) Google Scholar20Urbatsch I.L. Sankaran B. Weber J. Senior A.E. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site.J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Coupling of ATP hydrolysis in the NBDs to drug efflux from the drug-binding sites in the TMDs is mediated by ball-and-socket joints (21Jin M.S. Oldham M.L. Zhang Q. Chen J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.Nature. 2012; 490: 566-569Crossref PubMed Scopus (382) Google Scholar) at the NBD-TMD transmission interfaces. Ball-and-socket joints are intracellular loops (ICLs) connecting TM segments that contain a central intracellular helix (IH) that is in close contact to an NBD. Contacts between TMD1-NBD1 or TMD2-NBD2 are mediated by the first ICL of each TMD (ICL1 or ICL3). The second ICL of each TMD mediates TMD1-NBD2 (ICL2) or TMD2-NBD1 (ICL4) contacts. Contacts between the homologous halves (TMD1-NBD2 or TMD2-NBD1) appear to be particularly important for mammalian drug pumps. For example, the BCRP/ABCG2 drug pump lacks contacts equivalent to P-gp ICL1/ICL3 (22Rosenberg M.F. Bikadi Z. Chan J. Liu X. Ni Z. Cai X. Ford R.C. Mao Q. The human breast cancer resistance protein (BCRP/ABCG2) shows conformational changes with mitoxantrone.Structure. 2010; 18: 482-493Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In addition, we found that mutations to IH1 (ICL1) had little effect on activity or maturation while mutations to IH2 (ICL2) suggested the presence of a hydrophobic interface that was highly sensitive to changes (23Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.J. Biol. Chem. 2013; 288: 20326-20333Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The presence of such an interface was unexpected since modeling studies suggested that charged residues would play a dominant role (24Pajeva I.K. Hanl M. Wiese M. Protein contacts and ligand binding in the inward-facing model of human P-glycoprotein.ChemMedChem. 2013; 8: 748-762Crossref PubMed Scopus (25) Google Scholar). Hypersensitivity to mutation at the IH2/NBD2 transmission interface compared with the IH1/NBD1 interface might explain the differences in the maturation of NBD2 deletion mutants of P-gp and CFTR (25Loo T.W. Bartlett M.C. Shi L. Clarke D.M. Corrector-mediated rescue of misprocessed CFTR mutants can be reduced by the P-glycoprotein drug pump.Biochem. Pharmacol. 2012; 83: 345-354Crossref PubMed Scopus (17) Google Scholar). Deletion of NBD2 inhibits folding and trafficking of P-gp but not CFTR although both are structurally similar ABC proteins (26Wang Y. Loo T.W. Bartlett M.C. Clarke D.M. Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones.Mol. Pharmacol. 2007; 71: 751-758Crossref PubMed Scopus (66) Google Scholar). It is possible that ICL2/ICL3 interactions with NBD2 are critical for P-gp maturation and the protein is destabilized when NBD2 is deleted. Understanding P-gp domain-domain interactions will also be of importance when extrapolated to folding defects of other mutant ABC proteins associated with disease and in the design of methods to counteract their effects. It has been shown that in processing mutations of P-gp or CFTR trap the proteins as partially folded intermediates with incomplete NBD-TMD interactions (7Loo T.W. Bartlett M.C. Clarke D.M. Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).J. Biol. Chem. 2008; 283: 28190-28197Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In this study, we tested whether the NBD2 transmission interface was particularly critical for P-gp assembly and activity by comparing the effects of mutations at the NBD1-TMD and NBD2-TMD interfaces. We report that the interfaces are asymmetric since P-gp maturation was only sensitive to mutations at the NBD2 interface. The results suggest that the NBD2-TMD interface is a linchpin for assembly and repair of ABC proteins. Mutations were introduced into the wild-type or Cys-less P-gp cDNAs containing the A52-epitope or 10-histidine tags (27Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.Biochem. Biophys. Res. Commun. 2010; 395: 436-440Crossref PubMed Scopus (54) Google Scholar) by site-directed mutagenesis as described by Kunkel (28Kunkel T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). In most cases, residues in ICL1 (residues 146–177), ICL2 (residues 250–277), ICL3 (residues 787–818) or ICL4 (residues 893–920) were replaced with alanines to test for effects on maturation. Exceptions were that alanines were replaced with leucine, glycines were replaced with valine, and leucines or valines were replaced with serine as performed previously (23Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.J. Biol. Chem. 2013; 288: 20326-20333Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29Loo T.W. Clarke D.M. Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 30Loo T.W. Clarke D.M. Mutational analysis of ABC proteins.Arch. Biochem. Biophys. 2008; 476: 51-64Crossref PubMed Scopus (74) Google Scholar). Mutants were constructed to contain an A52 epitope tag at their C-terminal ends for use in whole cell immunoblot assays (29Loo T.W. Clarke D.M. Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar). The presence of the epitope tag distinguished the mutant proteins from any endogenous P-gp. P-gp contains three N-linked glycosylation sites can be used to monitor maturation of human P-gp from an immature 150 kDa protein to a mature 170 kDa protein. P-gp cDNA was modified to contain a 10-histidine tag at the COOH-terminal end to facilitate purification of the expressed protein by nickel-chelate chromatography (31Loo T.W. Clarke D.M. Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities.J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The ICL mutant cDNAs were transiently expressed in HEK 293 cells by a calcium phosphate precipitation approach as described previously (7Loo T.W. Bartlett M.C. Clarke D.M. Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).J. Biol. Chem. 2008; 283: 28190-28197Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, 10 μl of 2.5 m CaCl2 was added to 90 μl H2O containing 2 μg of DNA followed by addition of 100 μl of BES solution (50 mm N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 280 mm NaCl and 1.5 mm Na2HPO4, pH 6.96). After 10 min at room temperature, 4 ml of HEK 293 cells (about 100,000 cells/ml) in Dulbecco's modified Eagle's medium (DMEM) with high glucose (supplemented with nonessential amino acids, 4 mm l-glutamine, 10 IU/ml penicillin, 10 μg/ml streptomycin, and 10% (v/v) bovine calf serum) was added and 1.5 ml of the mixtures were added to duplicate well of 6-well culture plates. After 5 h at 37 °C, the medium was replaced with fresh medium with or without 0.5 μm tariquidar. About 16 h later, the cells were harvested, washed with PBS, and cell pellets suspended in 150 μl of 2× SDS sample buffer (125 mm Tris-HCl, pH 6.8, 4% (w/v) SDS, 4% (v/v) 2-mercaptoethanol containing 25 mm EDTA. Samples were applied to 6.5% SDS-PAGE gels (minigels, 1.5 mm spacers, 15 wells). The gels were electroblotted onto a sheet of nitrocellulose and P-gp proteins detected using A52 monoclonal antibody, horseradish peroxidase conjugated anti-mouse secondary antibody, and enhanced chemiluminescence. The signals were imaged and levels of mature (170 kDa) P-gp relative to total P-gp (mature 170 kDa plus immature 150 kDa) determined using ChemidocTM XRS+ with Image LabTM software (Bio-Rad Lab. Inc., Mississauga, Ontario). An equivalent amount of the sample was loaded onto 10% (v/v) SDS-PAGE gels and subjected to immunoblot analysis with a monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GADPH) (internal control). HEK 293 cells were plated onto fifty plates (10-cm diameter) and transfected with the cDNA of the histidine-tagged P-gp mutant when about 50% confluent. After 16 h at 37 °C, the medium was replaced with fresh medium containing 5 μm cyclosporin A. Cyclosporin A is a substrate of P-gp and acts as a potent pharmacological chaperone in promoting maturation and yield of P-gp (9Loo T.W. Clarke D.M. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Cyclosporin A rather than tariquidar was used to rescue the mutants for purification because it is considerably less expensive when used in scaled up experiments. The cells were harvested after another 24 h at 37 °C and washed three times with phosphate-buffered saline (PBS, pH 7.4). The cells were then suspended in PBS and solubilized at 4 °C by addition of one volume (0.75 ml) of PBS containing 2% (w/v) n-dodecyl-β-d-maltoside (Anatrace Inc., Maumee, OH). After 10 min at 4 °C, insoluble material was removed by centrifugation at 16 000 × g for 15 min at 4 °C. DNA in the supernatant was removed by passage through a DNA miniprep microfuge column (Bio Basic Canada Inc., Markham, ON). The flow-through material was then applied onto a nickel spin column (Ni-NTA, Qiagen, Mississauga, ON) that had been pre-equilibrated with buffer A containing 50 mm NaPO4, pH 8.0, 500 mm NaCl, 50 mm imidazole and 20% (v/v) glycerol and 0.1% (w/v) n-dodecyl-β-d-maltoside. The column was then washed twice with 0.6 ml buffer B containing 10 mm Tris-HCl, pH 7.5, 500 mm NaCl, 80 mm imidazole, pH 7.0, 20% (v/v) glycerol and 0.1% (w/v) n-dodecyl-β-d-maltoside and twice with 0.6 ml buffer C (buffer B containing 50 mm imidazole). Histidine-tagged P-gp was then eluted with 0.2 ml of buffer B but containing 300 mm imidazole. Recovery of P-gp was monitored by immunoblot analysis with rabbit anti-P-gp polyclonal antibody (32Loo T.W. Clarke D.M. P-glycoprotein. Associations between domains and between domains and molecular chaperones.J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). A sample of the isolated histidine-tagged P-gp (about 100 ng) was mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S, Sigma) that had been washed and suspended in TBS. ATPase activity (33Chifflet S. Torriglia A. Chiesa R. Tolosa S. A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases.Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (417) Google Scholar) was measured in the presence of 0.4 mm verapamil. Cys-less P-gp or the ICL1/ICL4 double cysteine mutants I160C/F904C or F163C/R905C were transiently expressed in HEK 293 cells at reduced temperature (30 °C) to promote maturation. The P-gps were isolated by nickel-chelate chromatography. The isolated P-gps were incubated 20 °C for 10 min in the presence or absence of 0.5 mm copper phenanthroline (oxidant to promote disulfide bond formation). EDTA was then added to a final concentration of 2 mm. Immunoblot analysis and assay of ATPase activity were performed as described above. The NBDs of P-gp are linked to the TMDs by four ICLs (Fig. 1, A and B). The IH located in the middle of the ICLs interacts with the NBDs to form ball-and-socket joints (21Jin M.S. Oldham M.L. Zhang Q. Chen J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.Nature. 2012; 490: 566-569Crossref PubMed Scopus (382) Google Scholar). The NBD1 transmission interface is linked to TMD1 and TMD2 by ICL1 and ICL4, respectively. The NBD2 transmission interface is linked to TMD1 and TMD2 by ICL2 and ICL3, respectively. The transmission interfaces might play critical roles to promote folding of P-gp into a native structure. P-gp is initially synthesized in the endoplasmic reticulum to yield a protease-sensitive loosely folded protein with incomplete packing of the TM segments and incomplete domain-domain interactions (34Loo T.W. Bartlett M.C. Clarke D.M. Processing Mutations Located throughout the Human Multidrug Resistance P-glycoprotein Disrupt Interactions between the Nucleotide Binding Domains.J. Biol. Chem. 2004; 279: 38395-38401Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 35Loo T.W. Clarke D.M. Superfolding of the Partially Unfolded Core-glycosylated Intermediate of Human P-glycoprotein into the Mature Enzyme Is Promoted by Substrate- induced Transmembrane Domain Interactions.J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar36Loo T.W. Clarke D.M. The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.Faseb J. 1999; 13: 1724-1732Crossref PubMed Scopus (81) Google Scholar). P-gp then matures into a compact protease-resistant native conformation that leaves the endoplasmic reticulum for addition of complex carbohydrate in the Golgi and trafficking to the plasma membrane (36Loo T.W. Clarke D.M. The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.Faseb J. 1999; 13: 1724-1732Crossref PubMed Scopus (81) Google Scholar). Removal of the NBDs inhibits maturation of P-gp. P-gp is different from CFTR because deletion of NBD2 only inhibits P-gp maturation (26Wang Y. Loo T.W. Bartlett M.C. Clarke D.M. Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones.Mol. Pharmacol. 2007; 71: 751-758Crossref PubMed Scopus (66) Google Scholar). This indicated that the NBD2-TMD interface might play a particularly important role in the mechanism of P-gp folding. Truncation mutants lacking NBD2 or both NBDs are trapped in the endoplasmic reticulum in protease-sensitive loosely folded conformations (37Loo T.W. Clarke D.M. The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A mutational approach was used to test if residues in the ICLs at the NBD2 transmission interface played more important roles in maturation of P-gp compared with residues in the ICLs at the NBD1 interface. Maturation of P-gp can readily be monitored in whole cell assays as the protein contains three N-glycosylation sites in the extracellular loop connecting TM segments 1 and 2 (Fig. 1A). The protein is initially synthesized as a 150 kDa core-glycosylated protein. If the protein correctly folds into a compact structure it can exit the endoplasmic reticulum for modification of the carbohydrate in the Golgi to yield a 170 kDa mature protein. Accordingly, 84 mutants were constructed that contained point mutations to residues in IH4/ICL4 and residues in ICL1, ICL2, and ICL3 flanking IH1, IH2, and IH3, respectively (segments of about 30 residues). The 36 IH1, IH2, and IH3 mutants constructed in previous studies (23Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.J. Biol. Chem. 2013; 288: 20326-20333Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 38Loo T.W. Clarke D.M. Locking Intracellular Helices 2 and 3 Together Inactivates Human P-glycoprotein.J. Biol. Chem. 2014; 289: 229-236Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) were included for comparison. Mutations were made to amino acids flanking each IH as it had been reported that some of these residues contributed to NBD-TMD interactions in the crystal structure of P-gp from Caenorhabditis elegans (21Jin M.S. Oldham M.L. Zhang Q. Chen J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.Nature. 2012; 490: 566-569Crossref PubMed Scopus (382) Google Scholar). In general, residues were replaced with alanine as it has a small side chain. Exceptions were that alanines were replaced with leucine, glycines were replaced with valine, and leucines or valines were replaced with serine as performed previously (23Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.J. Biol. Chem. 2013; 288: 20326-20333Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29Loo T.W. Clarke D.M. Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 30Loo T.W. Clarke D.M. Mutational analysis of ABC proteins.Arch. Biochem. Biophys. 2008; 476: 51-64Crossref PubMed Scopus (74) Google Scholar). Mutants were transiently expressed in HEK 293 cells for about 16 h and whole cell SDS extracts were subjected to immunoblot analysis to determine the steady state levels of mature and immature forms of P-gp. Examples of the effects of the mutations compared with wild-type P-gp are shown in Fig. 2A. Wild-type P-gp showed efficient maturation as about 80% of the protein was present as the 170 kDa mature form of the protein. The remainder of the protein was the 150 kDa immature P-gp. We previously showed that the 150 kDa protein was core-glycosylated as it was sensitive to endoglycosidases H and F (39Loo T.W. Clarke D.M. A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein." @default.
• W218221756 created "2016-06-24" @default.
• W218221756 creator A5042609718 @default.
• W218221756 creator A5060459035 @default.
• W218221756 date "2015-07-01" @default.
• W218221756 modified "2023-10-12" @default.
• W218221756 title "The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein" @default.
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