Glutathione-dependent Binding of a Photoaffinity Analog of Agosterol A to the C-terminal Half of Human Multidrug Resistance Protein

Abstract

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD 0 ), which is connected to a P-glycoprotein-like core region (⌬MRP) by a cytoplasmic linker domain zero (L 0 ).It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD 0 and L 0 are not known. We synthesized [125 I]11-azidophenyl agosterol A ([ 125 I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C 932-1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C 4 . Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L 0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L 0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (Pgp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1-4). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5), they share only 15% amino acid sequence identity (6). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD 0 ), which is connected to the P-gp-like core (⌬MRP) by a cytoplasmic linker domain zero (L 0 ) (7, 8).The precise roles of TMD 0 and L 0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9, 10), there is compelling evidence that they function very differently in drug transport (6, 11). P-gp confers multidrug resistance by directly binding and transporting unmodified drugs (12, 13). MRP1, however, is an active transporter of amphiphilic conjugated organic anions, including a number of compounds conjugated with GSH, glucuronide, and sulfate. To date, leukotriene C 4 (LTC 4 ) is the best substrate for MRP1 (14). It has been reported that GSH plays a critical role in MRP1-mediated MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine su...

The MRP family of drug efflux pumps

“…LTC4 binding sites were mapped to a region encompassing MSD1-NBD1 and to TM14-17, and LY474776 binding was mapped to TM16-17. In addition, agosterol A, a polyhydroxylated sterol acetate that is a potent glutathione-dependent inhibitor, was also reported to crosslink TM17 (Ren et al, 2001(Ren et al, , 2002. The results of site-directed mutagenesis studies support the involvement of TM14 and TM17 in MRP1 activity, and have also implicated residues in TM6 (Ito et al, 2001b;Zhang et al, 2001aZhang et al, , b, 2002Haimeur et al, 2002;Ren et al, 2002).…”

“…Glutathione-dependent binding of agosterol A to the COOH half of the protein required the presence of the L0 domain, implicating the latter region in glutathione binding (Ren et al, 2001). MRP1 can be crosslinked with glutathione photoaffinity reagents (Ciaccio et al, 1996), and studies using these probes have provided more direct information on sites of glutathione interaction.…”

Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione

“…Both iodinated compounds have been shown to cross-link preferentially to proteolytic peptides that contain either TM helices 10 and 11 or helices 16 and 17 (31). A third non-physiological compound, an azido derivative of the marine sponge polyhydroxylated sterol acetate, agosterol-A (AG-A), has also been used to photolabel MRP1 (34). In contrast to the other compounds, binding of AG-A is GSH dependent, and labeling is restricted to a site in the COOH-proximal half of the protein (34).…”

“…As an alternative approach to identifying regions of the protein directly involved in substrate binding, MRP1 has been photolabeled by its known high affinity physiological substrate LTC 4 as well as by compounds that are less well characterized with respect to binding and transport (10,12,27,(31)(32)(33)(34). Although these compounds compete with LTC 4 for binding to MRP1, their affinities appear to be considerably lower, and they also interact with the distantly related P-glycoprotein.…”

The Role of the Conserved Glycines of ATP-binding Cassette Signature Motifs of MRP1 in the Communication between the Substrate-binding Site and the Catalytic Centers

“…On one hand, the sites interacting with the drug substrates are presumably harbored in the transmembrane domains (6 -8); however, recent data suggest that the intracellular linkers L0 and L1 may also play role in binding drug substrates (9,10). On the other hand, the ATP hydrolysis, which energizes the transport, takes place within the ABC domains.…”