This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Azzaria, M Articles by Gros, P Search for Related Content PubMed PubMed Citation Articles by Azzaria, M Articles by Gros, P

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

ABSTRACT

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

This article has been cited by other articles:

• Farzaneh, T., Tinker, A. (2008). Differences in the mechanism of metabolic regulation of ATP-sensitive K+ channels containing Kir6.1 and Kir6.2 subunits. Cardiovasc Res 0: cvn138v2-11 [Abstract] [Full Text]  
• Linton, K. J. (2007). Structure and Function of ABC Transporters. Physiology 22: 122-130 [Abstract] [Full Text]  
• Schroeder, L. K., Kremer, S., Kramer, M. J., Currie, E., Kwan, E., Watts, J. L., Lawrenson, A. L., Hermann, G. J. (2007). Function of the Caenorhabditis elegans ABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle. Mol. Biol. Cell 18: 995-1008 [Abstract] [Full Text]  
• Zhao, Q., Chang, X.-b. (2004). Mutation of the Aromatic Amino Acid Interacting with Adenine Moiety of ATP to a Polar Residue Alters the Properties of Multidrug Resistance Protein 1. J. Biol. Chem. 279: 48505-48512 [Abstract] [Full Text]  
• Tombline, G., Bartholomew, L. A., Urbatsch, I. L., Senior, A. E. (2004). Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly. J. Biol. Chem. 279: 31212-31220 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2004). Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein. J. Biol. Chem. 279: 18232-18238 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2004). Disulfide Cross-linking Analysis Shows That Transmembrane Segments 5 and 8 of Human P-glycoprotein Are Close Together on the Cytoplasmic Side of the Membrane. J. Biol. Chem. 279: 7692-7697 [Abstract] [Full Text]  
• Tombline, G., Bartholomew, L., Gimi, K., Tyndall, G. A., Senior, A. E. (2004). Synergy between Conserved ABC Signature Ser Residues in P-glycoprotein Catalysis. J. Biol. Chem. 279: 5363-5373 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2003). Methanethiosulfonate Derivatives of Rhodamine and Verapamil Activate Human P-glycoprotein at Different Sites. J. Biol. Chem. 278: 50136-50141 [Abstract] [Full Text]  
• Rao, U. S., Nuti, S. L. (2003). Identification of Two Different States of P-glycoprotein in Its Catalytic Cycle: ROLE OF THE LINKER REGION IN THE TRANSITION BETWEEN THESE TWO STATES. J. Biol. Chem. 278: 46576-46582 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2003). Simultaneous Binding of Two Different Drugs in the Binding Pocket of the Human Multidrug Resistance P-glycoprotein. J. Biol. Chem. 278: 39706-39710 [Abstract] [Full Text]  
• Payen, L. F., Gao, M., Westlake, C. J., Cole, S. P. C., Deeley, R. G. (2003). Role of Carboxylate Residues Adjacent to the Conserved Core Walker B Motifs in the Catalytic Cycle of Multidrug Resistance Protein 1 (ABCC1). J. Biol. Chem. 278: 38537-38547 [Abstract] [Full Text]  
• Samanta, S., Ayvaz, T., Reyes, M., Shuman, H. A., Chen, J., Davidson, A. L. (2003). Disulfide Cross-linking Reveals a Site of Stable Interaction between C-terminal Regulatory Domains of the Two MalK Subunits in the Maltose Transport Complex. J. Biol. Chem. 278: 35265-35271 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2003). Permanent Activation of the Human P-glycoprotein by Covalent Modification of a Residue in the Drug-binding Site. J. Biol. Chem. 278: 20449-20452 [Abstract] [Full Text]  
• Maki, N., Hafkemeyer, P., Dey, S. (2003). Allosteric Modulation of Human P-glycoprotein. INHIBITION OF TRANSPORT BY PREVENTING SUBSTRATE TRANSLOCATION AND DISSOCIATION. J. Biol. Chem. 278: 18132-18139 [Abstract] [Full Text]  
• Loo, T. W., Bartlett, M. C., Clarke, D. M. (2003). Drug Binding in Human P-glycoprotein Causes Conformational Changes in Both Nucleotide-binding Domains. J. Biol. Chem. 278: 1575-1578 [Abstract] [Full Text]  
• Ozvegy, C., Varadi, A., Sarkadi, B. (2002). Characterization of Drug Transport, ATP Hydrolysis, and Nucleotide Trapping by the Human ABCG2 Multidrug Transporter. MODULATION OF SUBSTRATE SPECIFICITY BY A POINT MUTATION. J. Biol. Chem. 277: 47980-47990 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2002). Location of the Rhodamine-binding Site in the Human Multidrug Resistance P-glycoprotein. J. Biol. Chem. 277: 44332-44338 [Abstract] [Full Text]  
• Fetsch, E. E., Davidson, A. L. (2002). From the Cover: Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter. Proc. Natl. Acad. Sci. USA 99: 9685-9690 [Abstract] [Full Text]  
• Borchers, C., Boer, R., Klemm, K., Figala, V., Denzinger, T., Ulrich, W.-R., Haas, S., Ise, W., Gekeler, V., Przybylski, M. (2002). Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry. Mol. Pharmacol. 61: 1366-1376 [Abstract] [Full Text]  
• Matsushita, K., Kinoshita, K., Matsuoka, T., Fujita, A., Fujikado, T., Tano, Y., Nakamura, H., Kurachi, Y. (2002). Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels. Circ. Res. 90: 554-561 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2002). Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site. Proc. Natl. Acad. Sci. USA 99: 3511-3516 [Abstract] [Full Text]  
• Davidson, A. L. (2002). Mechanism of Coupling of Transport to Hydrolysis in Bacterial ATP-Binding Cassette Transporters. J. Bacteriol. 184: 1225-1233 [Full Text]  
• Vigano, C., Julien, M., Carrier, I., Gros, P., Ruysschaert, J.-M. (2002). Structural and Functional Asymmetry of the Nucleotide-binding Domains of P-glycoprotein Investigated by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy. J. Biol. Chem. 277: 5008-5016 [Abstract] [Full Text]  
• Qian, Y.-M., Qiu, W., Gao, M., Westlake, C. J., Cole, S. P. C., Deeley, R. G. (2001). Characterization of Binding of Leukotriene C4 by Human Multidrug Resistance Protein 1. EVIDENCE OF DIFFERENTIAL INTERACTIONS WITH NH2- AND COOH-PROXIMAL HALVES OF THE PROTEIN. J. Biol. Chem. 276: 38636-38644 [Abstract] [Full Text]  
• Reimann, F., Ashcroft, F. M., Gribble, F. M. (2001). Structural Basis for the Interference Between Nicorandil and Sulfonylurea Action. Diabetes 50: 2253-2259 [Abstract] [Full Text]  
• Yoder, J. H., Han, M. (2001). Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis in Caenorhabditis elegans. Mol. Biol. Cell 12: 2921-2933 [Abstract] [Full Text]  
• Reimann, F., Gribble, F. M., Ashcroft, F. M. (2001). Differential Response of KATP Channels Containing SUR2A or SUR2B Subunits to Nucleotides and Pinacidil. Mol. Pharmacol. 58: 1318-1325 [Abstract] [Full Text]  
• Sharma, S., Davidson, A. L. (2000). Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis. J. Bacteriol. 182: 6570-6576 [Abstract] [Full Text]  
• Matsuoka, T., Matsushita, K., Katayama, Y., Fujita, A., Inageda, K., Tanemoto, M., Inanobe, A., Yamashita, S., Matsuzawa, Y., Kurachi, Y. (2000). C-Terminal Tails of Sulfonylurea Receptors Control ADP-Induced Activation and Diazoxide Modulation of ATP-Sensitive K+ Channels. Circ. Res. 87: 873-880 [Abstract] [Full Text]  
• Holloway, S. L. (2000). CHL1 is a nuclear protein with an essential ATP binding site that exhibits a size-dependent effect on chromosome segregation. Nucleic Acids Res 28: 3056-3064 [Abstract] [Full Text]  
• Kwan, T., Loughrey, H., Brault, M., Gruenheid, S., Urbatsch, I. L., Senior, A. E., Gros, P. (2000). Functional Analysis of a Tryptophan-Less P-glycoprotein: A Tool for Tryptophan Insertion and Fluorescence Spectroscopy. Mol. Pharmacol. 58: 37-47 [Abstract] [Full Text]  
• Mitsuhashi, N., Miki, T., Senbongi, H., Yokoi, N., Yano, H., Miyazaki, M., Nakajima, N., Iwanaga, T., Yokoyama, Y., Shibata, T., Seino, S. (2000). MTABC3, a Novel Mitochondrial ATP-binding Cassette Protein Involved in Iron Homeostasis. J. Biol. Chem. 275: 17536-17540 [Abstract] [Full Text]  
• Gribble, F. M., Reimann, F., Ashfield, R., Ashcroft, F. M. (2000). Nucleotide Modulation of Pinacidil Stimulation of the Cloned KATP Channel Kir6.2/SUR2A. Mol. Pharmacol. 57: 1256-1261 [Abstract] [Full Text]  
• Gao, M., Cui, H.-R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P. C., Deeley, R. G. (2000). Comparison of the Functional Characteristics of the Nucleotide Binding Domains of Multidrug Resistance Protein 1. J. Biol. Chem. 275: 13098-13108 [Abstract] [Full Text]  
• Matsuo, M., Kioka, N., Amachi, T., Ueda, K. (1999). ATP Binding Properties of the Nucleotide-binding Folds of SUR1. J. Biol. Chem. 274: 37479-37482 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1999). Identification of Residues in the Drug-binding Domain of Human P-glycoprotein. ANALYSIS OF TRANSMEMBRANE SEGMENT 11 BY CYSTEINE-SCANNING MUTAGENESIS AND INHIBITION BY DIBROMOBIMANE. J. Biol. Chem. 274: 35388-35392 [Abstract] [Full Text]  
• Lerner-Marmarosh, N., Gimi, K., Urbatsch, I. L., Gros, P., Senior, A. E. (1999). Large Scale Purification of Detergent-soluble P-glycoprotein from Pichia pastoris Cells and Characterization of Nucleotide Binding Properties of Wild-type, Walker A, and Walker B Mutant Proteins. J. Biol. Chem. 274: 34711-34718 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1999). 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. 274: 24759-24765 [Abstract] [Full Text]  
• GADSBY, D. C., NAIRN, A. C. (1999). Control of CFTR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79: 77-107 [Abstract] [Full Text]  
• Celis, R. T. F., Leadlay, P. F., Roy, I., Hansen, A. (1998). Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System. J. Bacteriol. 180: 4828-4833 [Abstract] [Full Text]  
• Hrycyna, C. A., Ramachandra, M., Ambudkar, S. V., Ko, Y. H., Pedersen, P. L., Pastan, I., Gottesman, M. M. (1998). Mechanism of Action of Human P-glycoprotein ATPase Activity. PHOTOCHEMICAL CLEAVAGE DURING A CATALYTIC TRANSITION STATE USING ORTHOVANADATE REVEALS CROSS-TALK BETWEEN THE TWO ATP SITES. J. Biol. Chem. 273: 16631-16634 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1998). 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. 273: 14671-14674 [Abstract] [Full Text]  
• Szabo, K., Welker, E., Bakos, E., Muller, M., Roninson, I., Varadi, A., Sarkadi, B. (1998). Drug-stimulated Nucleotide Trapping in the Human Multidrug Transporter MDR1. COOPERATION OF THE NUCLEOTIDE BINDING DOMAINS. J. Biol. Chem. 273: 10132-10138 [Abstract] [Full Text]  
• Egner, R., Rosenthal, F. E., Kralli, A., Sanglard, D., Kuchler, K. (1998). Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter. Mol. Biol. Cell 9: 523-543 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1997). Identification of Residues in the Drug-binding Site of Human P-glycoprotein Using a Thiol-reactive Substrate. J. Biol. Chem. 272: 31945-31948 [Abstract] [Full Text]  
• Dey, S., Ramachandra, M., Pastan, I., Gottesman, M. M., Ambudkar, S. V. (1997). Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc. Natl. Acad. Sci. USA 94: 10594-10599 [Abstract] [Full Text]  
• Ueda, K., Inagaki, N., Seino, S. (1997). MgADP Antagonism to Mg2+-independent ATP Binding of the Sulfonylurea Receptor SUR1. J. Biol. Chem. 272: 22983-22986 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1997). Drug-stimulated ATPase Activity of Human P-glycoprotein Requires Movement between Transmembrane Segments 6 and 12. J. Biol. Chem. 272: 20986-20989 [Abstract] [Full Text]  
• Ortiz, D. F., Pierre, M. V. St., Abdulmessih, A., Arias, I. M. (1997). A Yeast ATP-binding Cassette-type Protein Mediating ATP-dependent Bile Acid Transport. J. Biol. Chem. 272: 15358-15365 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1996). The Minimum Functional Unit of Human P-glycoprotein Appears to be a Monomer. J. Biol. Chem. 271: 27488-27492 [Abstract] [Full Text]  
• Dayan, G., Baubichon-Cortay, Hélčn., Jault, J.-M., Cortay, J.-C., Deléage, G., Di Pietro, A. (1996). Recombinant N-terminal Nucleotide-binding Domain from Mouse P-glycoprotein. J. Biol. Chem. 271: 11652-11658 [Abstract] [Full Text]  
• Müller, M., Bakos, Év., Welker, E., Váradi, A.ás, Germann, U. A., Gottesman, M. M., Morse, B. S., Roninson, I. B., Sarkadi, B.áz. (1996). Altered Drug-stimulated ATPase Activity in Mutants of the Human Multidrug Resistance Protein. J. Biol. Chem. 271: 1877-1883 [Abstract] [Full Text]  
• Urbatsch, I. L., Sankaran, B., Bhagat, S., Senior, A. E. (1995). Both P-glycoprotein Nucleotide-binding Sites Are Catalytically Active. J. Biol. Chem. 270: 26956-26961 [Abstract] [Full Text]  
• Sachs, C. W., Safa, A. R., Harrison, S. D., Fine, R. L. (1995). Partial Inhibition of Multidrug Resistance by Safingol Is Independent of Modulation of P-glycoprotein Substrate Activities and Correlated with Inhibition of Protein Kinase C. J. Biol. Chem. 270: 26639-26648 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1995). Covalent Modification of Human P-glycoprotein Mutants Containing a Single Cysteine in Either Nucleotide-binding Fold Abolishes Drug-stimulated ATPase Activity. J. Biol. Chem. 270: 22957-22961 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (1995). P-glycoprotein. J. Biol. Chem. 270: 21839-21844 [Abstract] [Full Text]  
• Beaudet, L., Gros, P. (1995). Functional Dissection of P-glycoprotein Nucleotide-binding Domains in Chimeric and Mutant Proteins. J. Biol. Chem. 270: 17159-17170 [Abstract] [Full Text]  
• Sharma, S., Rose, D. R. (1995). Cloning, Overexpression, Purification, and Characterization of the Carboxyl-terminal Nucleotide Binding Domain of P-glycoprotein. J. Biol. Chem. 270: 14085-14093 [Abstract] [Full Text]  
• Carson, M. R., Travis, S. M., Welsh, M. J. (1995). The Two Nucleotide-binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Have Distinct Functions in Controlling Channel Activity. J. Biol. Chem. 270: 1711-1717 [Abstract] [Full Text]  
• Sardini, A, Mintenig, G., Valverde, M., Sepulveda, F., Gill, D., Hyde, S., Higgins, C., McNaughton, P. (1994). Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling. J. Cell Sci. 107: 3281-3290 [Abstract]  
• Anderson, M., Welsh, M. (1992). Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains. Science 257: 1701-1704 [Abstract]  
• Gibson, A., Wagner, L., Collins, F., Oxender, D. (1991). A bacterial system for investigating transport effects of cystic fibrosis--associated mutations. Science 254: 109-111 [Abstract]  
• Loo, T. W., Clarke, D. M. (2000). Drug-stimulated ATPase Activity of Human P-glycoprotein Is Blocked by Disulfide Cross-linking between the Nucleotide-binding Sites. J. Biol. Chem. 275: 19435-19438 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2001). Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers. J. Biol. Chem. 276: 36877-36880 [Abstract] [Full Text]  
• Hou, Y.-x., Cui, L., Riordan, J. R., Chang, X.-b. (2000). Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1. J. Biol. Chem. 275: 20280-20287 [Abstract] [Full Text]  
• Urbatsch, I. L., Gimi, K., Wilke-Mounts, S., Senior, A. E. (2000). Conserved Walker A Ser Residues in the Catalytic Sites of P-glycoprotein Are Critical for Catalysis and Involved Primarily at the Transition State Step. J. Biol. Chem. 275: 25031-25038 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2000). Identification of Residues within the Drug-binding Domain of the Human Multidrug Resistance P-glycoprotein by Cysteine-scanning Mutagenesis and Reaction with Dibromobimane. J. Biol. Chem. 275: 39272-39278 [Abstract] [Full Text]  
• Lapinski, P. E., Neubig, R. R., Raghavan, M. (2001). Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding. J. Biol. Chem. 276: 7526-7533 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2001). Defining the Drug-binding Site in the Human Multidrug Resistance P-glycoprotein Using a Methanethiosulfonate Analog of Verapamil, MTS-verapamil. J. Biol. Chem. 276: 14972-14979 [Abstract] [Full Text]  
• Ketchum, C. J., Schmidt, W. K., Rajendrakumar, G. V., Michaelis, S., Maloney, P. C. (2001). The Yeast a-factor Transporter Ste6p, a Member of the ABC Superfamily, Couples ATP Hydrolysis to Pheromone Export. J. Biol. Chem. 276: 29007-29011 [Abstract] [Full Text]  
• Loo, T. W., Clarke, D. M. (2001). Cross-linking of Human Multidrug Resistance P-glycoprotein by the Substrate, Tris-(2-maleimidoethyl)amine, Is Altered by ATP Hydrolysis. EVIDENCE FOR ROTATION OF A TRANSMEMBRANE HELIX. J. Biol. Chem. 276: 31800-31805 [Abstract] [Full Text]  
• Karttunen, J. T., Lehner, P. J., Gupta, S. S., Hewitt, E. W., Cresswell, P. (2001). Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP). Proc. Natl. Acad. Sci. USA 98: 7431-7436 [Abstract] [Full Text]  
• Matsushita, K., Kinoshita, K., Matsuoka, T., Fujita, A., Fujikado, T., Tano, Y., Nakamura, H., Kurachi, Y. (2002). Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels. Circ. Res. 90: 554-561 [Abstract] [Full Text]