Nonequivalent Nucleotide Trapping in the Two Nucleotide Binding Folds of the Human Multidrug Resistance Protein MRP1

Abstract

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadateinduced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[␣-32 P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.Multidrug resistance of tumor cells is a major obstacle to cancer chemotherapy. This phenomenon is frequently associated with the expression of P-glycoprotein and multidrug resistance protein 1 (MRP1), 1 both of which are ATP binding cassette (ABC) proteins. P-glycoprotein and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from the cells before they reach their intracellular targets, thus conferring resistance to many structurally dissimilar anticancer drugs, such as the Vinca alkaloids, colchicine, actinomycin D, etoposide, taxol, and anthracyclines (1-5). However, the mechanism of transport for MRP1 could be different from that for P-glycoprotein, because the depletion of intracellular glutathione (GSH) by buthionine sulfoximine results in a complete reversal of resistance to anticancer drugs of some cell lines expressing MRP1 (6, 7), but buthionine sulfoximine has no effect on P-glycoprotein-mediated multidrug resistance. It has been reported that MRP1 transports GSH-S-conjugates such as leukotriene C 4 , glutathione disulfide (GSSG), and 2,4-dinitrophenyl-S-glutathione (8 -11) and that MRP1 mediates ATP-dependent transport of vincristine, daunorubicin, and etoposide in the presence of . From these findings, it has been postulated that MRP1 can actively cotransport GSH and unmodified xenobiotics as well as GSH-S-conjugates.We have reported that MRP1 in membrane from a human MRP1 cDNA transformant can be specifically photoaffinity labeled with 8-azido-[␣-32 P]ATP by vanadate-induced nucleotide trapping (15). Vanadate and Mg 2ϩ were required for trapping of nucleotide, and photoaffinity labeling was inhibited by the excess ADP as well as ATP. These results have suggested that a stable inhibitory complex MRP1⅐MgADP⅐Vi, an analog of the MRP1⅐MgADP⅐P i transition state complex, is formed in the presence of vanadate, as suggested for P-glycoprotein (16). Vanadate-induced nucleotide trapping in P-glycoprotein has been reported to be stimulated by the t...

ATP Binding, Not Hydrolysis, at the First Nucleotide-binding Domain of Multidrug Resistance-associated Protein MRP1 Enhances ADP·Vi Trapping at the Second Domain

“…Solutes (for example, anti-cancer drugs) are extruded out of cells by MRP1 protein in an ATP-dependent manner (7,14,37) by utilizing its two NBDs to bind and hydrolyze ATP. Both NBDs of MRP1 protein can bind nucleotides (30,31,33). However, the properties and the functions of the two NBDs do not seem to be equal as discussed in the Introduction.…”

“…First, modifications of the consensus Walker motifs in the two NBDs do not inactivate the protein completely and have different effects on solute transport (30 -32). Second, photoaffinity labeling experiments with 8-azido-ATP also revealed an asymmetry between NBD1 and NBD2, with NBD1 preferentially labeled by 8-N 3 [␥-32 P]ATP (30,31), whereas NBD2 trapped the nucleoside diphosphate hydrolysis product (30,31,33). Third, the ATP binding/hydrolysis sites of MRP1 seem not to be recruited in a random manner because photolabeling by the nonhydrolyzable 8-N 3 [␣- 32 P]AMP-PNP occurred predominately at NBD1 (34), and the NBD1 fragment was labeled predominantly with 8-N 3 [␣-…”

“…Therefore, how events at NBD1 and NBD2 of P-gp cooperate during drug transport is still not clear. Considerable evidence has accumulated indicating that the two NBDs of some other ATP-binding cassette (ABC) transporters, including the sulfonylurea receptor (SUR1) (26,27) (30,31,33). Third, the ATP binding/hydrolysis sites of MRP1 seem not to be recruited in a random manner because photolabeling by the nonhydrolyzable 8-N 3 [␣-…”

ATP-sensitive K channel channel/enzyme multimer: Metabolic gating in the heart

“…Further, in other SUR isoforms, potassium channel openers maintain MgADP binding, thereby prolonging channel activity following removal of the nucleoside diphosphate [62]. Conversely, intermediates in the ATPase cycle may define the affinity of bound ligands to SUR, as proposed for ATP hydrolysis in other ABC proteins [63,64]. Specifically, the slow off-rate of potassium channel openers in the presence of magnesium nucleotides [65] suggests that engagement in the posthydrolytic conformation may in turn stabilize potassium channel opener binding.…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…The transition-state complex formation can be followed by the so-called "nucleotide trapping" measurement. This also means photoaffinity labeling with azido-ATP but under hydrolytic conditions in the presence of a phosphate-mimicking anion, e.g., vanadate [43,53,105]. This anion stabilizes the transition-state complex (MRP1·Mg·ADP·P i ) by replacing the gamma phosphate and arrests the transporter in this state.…”