Mutational Analysis of Ionizable Residues Proximal to the Cytoplasmic Interface of Membrane Spanning Domain 3 of the Multidrug Resistance Protein, MRP1 (ABCC1)

Situ, Donna; Haimeur, Anass; Conseil, Gwenaëlle; Sparks, Kathryn E.; Zhang, Da Wei; Deeley, Roger G.; Cole, Susan P.C.

doi:10.1074/jbc.m403832200

Cited by 43 publications

(59 citation statements)

References 41 publications

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“…In addition, because MRP1 can also transport GSH alone or together with substrates such as vincristine (14), and assuming that nucleotides play a common mechanistic role in the transport of different substrates, then these findings could be construed to indicate the presence of two distinct GSH binding sites: one where GSH undergoes transport, and a second where GSH elicits allosteric effects on MRP1 without being translocated across the membrane. Such an idea has been proposed previously on the basis of mutagenesis experiments where the mutation of a single residue prevents GSH transport, yet GSH-stimulated estrone sulfate transport is only mildly affected (50,51), and vice versa (52), 5 but this remains to be directly proven.…”

Section: Discussionmentioning

confidence: 97%

Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)

Rothnie

Callaghan

Deeley

et al. 2006

Journal of Biological Chemistry

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Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (K d ) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

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Section: Discussionmentioning

confidence: 97%

Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)

Rothnie

Callaghan

Deeley

et al. 2006

Journal of Biological Chemistry

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“…Materials- [14,15,19, (25,26). 4 These basic residues were also localized in the atomic homology models of MRP1 derived by molecular dynamics simulations using the crystal structure of the bacterial lipid transporter MsbA as template (10).…”

Section: Methodsmentioning

confidence: 99%

“…Charged residues are black, and polar residues are shaded gray. The three basic residues mutated in the present study are indicated by asterisks, and a diamond denotes Arg 1131 mutated in a previous study (15).…”

Section: Kinetic Analysis Of [ 3 H]e 2 17␤gmentioning

confidence: 99%

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Functional Importance of Three Basic Residues Clustered at the Cytosolic Interface of Transmembrane Helix 15 in the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Conseil

Deeley

Cole

2006

Journal of Biological Chemistry

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The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg 1138 , Lys 1141 , and Arg 1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg 1138 and Arg 1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C 4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[ 32 P]ADP. Mutation of Lys 1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys 1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.

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“…In addition, site-directed mutagenesis studies have identified a number of mutation-sensitive amino acids with respect to transport and/or binding of LTC 4 . Thus, nonconservative (and in some cases, conservative) substitutions of certain residues located in or proximal to the cytosolic interface of TM6 ( (Haimeur et al, , 2004aCampbell et al, 2004;Koike et al, 2004;Situ et al, 2004;Zhang et al, 2004). However, it is not known whether these amino acids are in direct contact with LTC 4 or whether they have an indirect but critical role in maintaining the architecture of the LTC 4 binding site on the protein, or both.…”

Section: Analyses Of Mrp1mentioning

confidence: 99%

Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4 Binding Sites

Oleschuk

Mao

et al. 2005

Molecular Pharmacology

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Multidrug resistance in tumor cells may be caused by reduced drug accumulation resulting from expression of one or more proteins belonging to the ATP-binding cassette (ABC) transporter superfamily. In addition to their drug efflux properties, certain ABC proteins such as multidrug resistance protein 1 (MRP1) (ABCC1) mediate the ATP-dependent transport of a broad array of organic anions. The intrinsically photoreactive glutathione-conjugated cysteinyl leukotriene C 4 (LTC 4 ) is a high-affinity physiological substrate of MRP1 and is widely regarded as a model compound for evaluating the substrate binding and transport properties of wild-type and mutant forms of the transporter. In the present study, we have optimized high-level expression of recombinant human MRP1 in Pichia pastoris and developed a two-step purification scheme that results in purification of the transporter to Ͼ90% homogeneity.Peptide mapping by matrix-assisted laser desorption ionization/time of flight mass spectrometry of the peptides generated by in-gel protease digestions of purified underglycosylated MRP1 identified 96.7% of the MRP1 sequence with Ͼ98% coverage of its 17 transmembrane helices. Subsequent comparisons with mass spectra of MRP1 photolabeled with LTC 4 identified six candidate LTC 4 -modified peptide fragments that are consistent with the conclusion that the intracellular juxtamembrane positions of transmembrane helices 6, 7, 10, 17, and a COOH-proximal portion of the cytoplasmic loop that links the first and second membrane spanning domains are part of the LTC 4 binding site of the transporter. Our studies confirm the usefulness of mass spectrometry for analysis of mammalian polytopic membrane proteins and for identification of substrate binding sites of human MRP1.

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Mutational Analysis of Ionizable Residues Proximal to the Cytoplasmic Interface of Membrane Spanning Domain 3 of the Multidrug Resistance Protein, MRP1 (ABCC1)

Cited by 43 publications

References 41 publications

Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)

Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)

Functional Importance of Three Basic Residues Clustered at the Cytosolic Interface of Transmembrane Helix 15 in the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4 Binding Sites

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