The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics (original) (raw)
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Journal of Biological Chemistry, 2005
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.
Journal of Biological Chemistry, 2002
The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C 4 (LTC 4), 17-estradiol 17-(-D-glucuronide) (E 2 17G), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys 332 , His 335 , and Asp 336 , predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys 332 mutants had lost the ability to transport LTC 4 , and GSH transport was reduced; whereas E 2 17G, estrone 3-sulfate, and MTX transport were unaffected. E 2 17G transport was not inhibited by LTC 4 and could not be photolabeled with [ 3 H]LTC 4 , indicating that the MRP1-Lys 332 mutants no longer bound this substrate. Substitutions of MRP1-His 335 also selectively diminished LTC 4 transport and photolabeling but to a lesser extent. Kinetic analyses showed that V max (LTC 4) of these mutants was decreased but K m was unchanged. In contrast to the selective loss of LTC 4 transport in the Lys 332 and His 335 mutants, the MRP1-Asp 336 mutants no longer transported LTC 4 , E 2 17G, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys 332 , His 335 , and Asp 336 of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys 332 and His 335 in determining substrate specificity and of Asp 336 in overall transport activity suggests that such interactions are critical for the binding and transport of LTC 4 and other substrates of MRP1.
Biochemical and Biophysical Research Communications, 2004
Multidrug resistance in human tumour cells is often associated with increased expression of the 190 kDa multidrug resistance protein, MRP1, that belongs to the ATP-binding cassette superfamily of transport proteins. MRP1 is also an efficient transporter of many organic anions. In the present study, we have mapped the epitope of the MRP1-specific murine monoclonal antibody (MAb) MRPm5 to the decapeptide 1063 FFERTPSGNL 1072 located in the cytoplasmic loop (CL6) linking transmembrane helices 13 and 14 in the third membrane spanning domain of the protein. Several amino acids in the cytoplasmic loops of MRP1 have been reported to be important for its transport function; nevertheless, MAb MRPm5 does not inhibit vesicular uptake of the high affinity substrate leukotriene C 4. None of the other MRP1-reactive MAbs described to date map to CL6 of MRP1 which in turn enhances the utility of MAb MRPm5 for both clinical and experimental investigations of this transporter.
Intermediate Structural States Involved in MRP1-mediated Drug Transport
Journal of Biological Chemistry, 2002
Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC 4. The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.
The Structure of the Multidrug Resistance Protein 1 (MRP1/ABCC1)
Journal of Biological Chemistry, 2001
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH 2 terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are ϳ80 ؋ 100 Å. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to ϳ22-Å resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.
Journal of Biological Chemistry, 2004
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg 1046 and TM15 Arg 1131 did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg 1202 and Glu 1204 reduced protein expression by >80%; however, MRP1 expression was not affected when Arg 1202 and Glu 1204 were replaced with neutral or samecharge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg 1197 and TM17 Arg 1249 did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg 1197 or Arg 1249 were also transport-inactive and no longer bound leukotriene C 4. In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu 1204 serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.
Molecular Pharmacology, 2004
Multidrug resistance protein 1 (MRP1) belongs to the ATPbinding cassette superfamily of transport proteins. In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions. MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH 2-terminal MSD1. MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys 332 and Asp 336. We have now extended these studies and found that the samecharge TM6 mutant K332R, like the nonconservatively substituted Lys 332 mutants, exhibits a selective decrease in leukotriene C 4 (LTC 4) transport, associated with substantial changes in
Functional Multidrug Resistance Protein (MRP1) Lacking the N-terminal Transmembrane Domain
Journal of Biological Chemistry, 1998
The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD 0 ) connected to the core by a cytoplasmic linker (L 0 ). We have studied truncated MRP1 versions containing either the MDRlike core alone or the core plus linker L 0 , produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Fulllength MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C 4 and N-ethylmaleimide glutathione. In addition, leukotriene C 4 -stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L 0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD 0 region but still containing L 0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD 0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD 0 L 0 and the MRP1 minus TMD 0 L 0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD 0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.
FEBS letters, 2015
The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.