Stimulation of ATPase Activity of Purified Multidrug Resistance-associated Protein by Nucleoside Diphosphates

Abstract

Membrane vesicles prepared from cells expressing the multidrug resistance-associated protein (MRP) transport glutathione S-conjugates of hydrophobic substrates in an ATP dependent manner. Purified MRP possesses ATPase activity which can be further stimulated by anticancer drugs or leukotriene C4. However, the detailed relationship between ATP hydrolysis and drug transport has not been established. How the ATPase activity of MRP is regulated in the cell is also not known. In this report, we have examined the effects of different nucleotides on the ATPase activity of purified MRP. We have found that pyrimidine nucleoside triphosphates have little effect on enzymatic activity. In contrast, purine nucleotides dATP, dGTP, and adenosine 5-(␤,␥-imido)triphosphate function as competitive inhibitors. Somewhat unexpectedly, low concentrations of all the nucleoside diphosphates (NDPs) tested, except UDP, stimulate the ATPase activity severalfold. ADP or GDP at higher concentrations was inhibitory, reflecting NDP binding to the substrate site. On the other hand, the enhancement of hydrolysis at low NDP concentrations must reflect interactions with a separate site. Therefore, we postulate the presence of at least two types of nucleotide binding sites on the MRP, a catalytic site(s) to which ATP preferentially binds and is hydrolyzed and a regulatory site to which NDPs preferentially bind and stimulate hydrolysis. Interestingly, the stimulatory effects of drugs transported by MRP and NDPs are not additive, i.e. drugs are not able to further stimulate the NDP-activated enzyme. Hence, the two activation pathways intersect at some point. Since both nucleotide binding domains of MRP are likely to be required for drug stimulation of ATPase activity, the two sites that we postulate may also involve both domains.The multidrug resistance-associated protein (MRP) 1 gene was cloned from a multidrug-resistant cell line, H69AR, which was obtained by stepwise selection in increasing concentration of doxorubicin (1). A single open reading frame of 1531 amino acids was defined, and the protein was found to be a member of the ABC superfamily of transport systems (1) and to contain three membrane-associated domains and two nucleotide binding domains (2). As predicted, cells transfected with MRP cDNA become multidrug-resistant (3-10). Mutations of lysine residues in the Walker A motif of the first and second nucleotide binding domains of MRP decreased drug resistance levels compared with wild-type MRP (5), implying that MRP functions as an ATP-dependent drug pump. Its function has been studied widely using membrane vesicles prepared from MRP-expressing cells (11-23). It was found that anticancer drugs, such as vincristine, cannot be transported by MRP, unless glutathione is also present (13). Several natural substrates, such as the glutathione S-conjugate, LTC4 (11-13, 16, 20) and other conjugates including glutathione S-conjugated prostaglandin A1 and A2 (23), 17-␤-estradiol 17-␤-D-glucuronide (14), bile salt, and glucuronosylhyodeoxycholate...

Flavonoid Structure-Activity Studies Identify 6-Prenylchrysin and Tectochrysin as Potent and Specific Inhibitors of Breast Cancer Resistance Protein ABCG2

“…The R482 residue might, directly or indirectly, play a role in the coupling between energy supply and drug transport, and this effect would be altered upon mutation. A comparable activation or inhibition of ATP hydrolysis by modulators has been often reported for P-glycoprotein (42) and MRP1 (17,43).…”

Interactions of the Human Multidrug Resistance Proteins MRP1 and MRP2 with Organic Anions

“…The vanadate-sensitive ATPase activity of the multidrug transporter MDR1 has been shown to reflect the substrate interactions of this protein: transported substrates significantly (up to 3-to 6-fold compared with the baseline level) stimulated the ATPase activity, whereas non MDR1 substrates had no effect (see Sarkadi et al, 1992;Scarborough, 1995). In one study, Chang et al (1998) examined the vanadate-sensitive ATPase of the purified MRP1 protein and found stimulation by GS conjugates, although stimulation was weak (1.3-to 1.5-fold). In the present experiments, by using the high-level expression of human MRP1 and MRP2 in Sf9 cells, we examined the effects of various compounds on the ATPase activity of both proteins in a membrane environment.…”

SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator

“…To date, this has not been demonstrated. Although the K ATP channel has a relatively slow rate of ATP hydrolysis (100 nmol Pi min K1 mg K1 protein; Mikhailov et al 2005) compared with other ABC proteins, it nevertheless lies within the range of that reported for MRP1 (from 5 to 470 nmol Pi min K1 mg K1 protein; Chang et al 1998;Mao et al 1999). Thus, it is not inconceivable that it serves as a transporter of an as yet unidentified substrate.There is accumulating evidence, however, that SUR1 also plays a role in insulin granule exocytosis.…”