Anti‐cancer drugs and glutathione stimulate vanadate‐induced trapping of nucleotide in multidrug resistance‐associated protein (MRP)

Abstract

Multidrug resistance-associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP-dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate-induced trapping of 8-azido-ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti-cancer drugs VP-16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8-azido-[alpha-32P]ATP by the vanadate-trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP x MgADP x Vi, an analog of the MRP x MgADP x Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti-cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.

The MRP family of drug efflux pumps

“…The predominant working model is that agents such as vinca alkaloids and anthracyclines are cotransported with glutathione ( Figure 3b). This model is supported by studies showing that: (i) transport of vincristine and anthracyclines in membrane vesicle uptake assays is dependent upon glutathione (Loe et al, 1996b); (ii) etoposide stimulates glutathione extrusion in wild-type but not mrp1-deficient ES cells ; (iii) MRP1 can be photolabeled with glutathione analogs (Ciaccio et al, 1996); (iv) glutathione stimulates vanadate-induced trapping of 8-azido-ATP (Taguchi et al, 1997);and (v) glutathione derivatives lacking a free sulfhydryl group can support MRP1-mediated transport . Alternative possibilities involving allosteric interactions are also under consideration.…”

Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export

“…We propose a working model in which MRP2 has two drug-binding sites: one with a relatively high affinity for GSH (G-site) and a low affinity for drug, and one with a relatively high affinity for drug and a low affinity for GSH (D-site). The presence of a GSH-binding site is not unlikely as Taguchi et al (1997) have shown by vanadate trapping experiments that GSH can bind to MRP1, and Bakos et al (2000) have found GSH-induced ATP-ase activity in MRP1-and MRP2-containing membrane vesicles. We think that both binding sites are occupied by GSH in the absence of drugs, resulting in a slow export of GSH (Paulusma et al, 1999).…”

Structure and mechanism of ATP-dependent phospholipid transporters

“…Generally speaking the mechanism by which both proteins hydrolyze ATP is analogous, as can be inferred by the fact that both types of proteins are sensitive to vanadate [130,131], which mimics the transition state of ATP during hydrolysis [132].…”