The ABCC/MRP subfamily
Numerous xenobiotics are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. These transport processes are mediated by specific transport proteins, by multidrug resistance proteins (MRPs). MRPs contribute to drug resistance in cancer cells that is the major obstacle of successful chemotherapy. Up to now, nine members of the human MRP subfamily have been identified, they are members of the ATP-binding cassette (ABC) superfamily. Our aim is to study the function, the structure and the molecular mechanism of human MRP proteins.

Structure and domains of MRPs: ABC proteins share the basics of molecular architecture: they are composed of the combinations of transmembrane domains (TMD) and ABC (ATP-Binding Cassette) domains. It is a general view that the minimal functional requirement of an active ABC-transporter is the presence of two TMDs and two ABCs. Our group was the first to demonstrate that in the members of MRP subfamily the core structure (TMD1,ABC1,TMD2,ABC2) is extended with an N-terminal transmembrane domain (TMD0), including 5 transmembrane helixes, and a connecting cytoplasmic loop (L0) [1,2]. This way, we have established the correct membrane topology model and domain arrangement of the members of the MRP family. Analysis of subsequently identified human MRP proteins indicated that not all of them possess TMD0 domain: MRP4,5,8,9 lack the TMD0 domain but contain the L0, whereas MRP1,2,3,6,7 possess both domains. We have discovered that the TMD0 domain is dispensable for function of human MRP1, as an truncated mutant that lack this domain is functional with respect to transport activity, assuming basolateral localization in polarized cells [3]. However, extending the deletion to L0 abrogates the activity of the pump, indicating that L0 domain is essential for function. We have shown that the L0 interacts with the core region of MRP1 [4].


Mechanism of function: Transport of substrates is tightly coupled to ATP-hydrolysis in ABC transporters. A unique feature of ABC proteins that they are composed of two ABC domains where ATP binding and hydrolysis occur. The two cooperating ABC domains form two composite active sites. We have examined the details of the catalytic cycle in MRP1, major multidrug transporter. We have introduced a novel experimental strategy based on vanadate-induced cleavage of the polypeptide chain into the MRP field and we have distinguished a low and a high affinity catalytic site. We have detected two different allosteric effects during the MRP1 ATPase catalytic cycle and found that they control two consecutive steps of the catalytic cycle. Nucleotide binding to the low affinity site accelerates the prehydrolytic intermediate formation in the other catalytic centre, while interaction of the transporter with its transported substrates stimulates a later reaction of the hydrolytic cycle, the formation of the posthydrolytic intermediate, which could be detected in both catalytic sites [5].

The ABC units harbor two consensus polypeptide sequences, the Walker A and Walker B, which are present in many ATP-binding proteins. In addition, in all ABC transporters a highly conserved polypeptide motif, the signature region (LSGGQ), is found between the two Walker motifs, which is diagnostic for the whole superfamily. We demonstrated that conserved glycine residues in the fourth position of the ABC signature regions in MRP1 are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates [6].


The physiological/pharmacological function of MRPs: Human MRP1 protein, the first member of the MRP family, has been demonstrated to function as a pump for cytostatic agents, conferring resistance to a broad range of anti-cancer drugs. This transporter is a ubiquitous glutathione conjugate transporter [7]. MRP2 protein, which localizes to the apical surface of hepatocytes, is a canalicular efflux pump and plays a crucial role in the biliary transport of anionic conjugates. Our results suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions across the apical membrane of the proximal tubules of the kidney. MRP3 protein, which localizes to the basolateral surface of hepatocytes, may function as a “backup” transporter for amphipatic conjugates in cholestatic conditions. It may have a role in the detoxification of hepatocytes by extruding bile acids and other conjugates into sinusoidal blood. Although the substrate selectivity of human MRP1, MRP2 and MRP3 overlaps, we and others have found differences in the kinetic parameters and modulation of conjugate transport by these transporters [8,9]. Our laboratory demonstrated that MRP6 protein do not transport glucuronide conjugates, but glutathione conjugates [10], MRPs lacking the TMD0 domain has characteristic transport features. In contrast to other MRPs, MRP4, MRP5, MRP8 were shown to transport cyclic nucleotides and confer resistance to certain nucleotide analogues.

References:

  1. Bakos É, Hegedűs T, Holló Z, Welker E, Tusnády GE, Zaman GJ, Flens MJ, Váradi A and Sarkadi B (1996)
    Membrane topology and glycosylation of the human multidrug resistance-associated protein.
    J Biol Chem 271, 12322-6 [PubMed]
  2. Tusnády GE, Bakos É, Váradi A and Sarkadi B (1997)
    Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters.
    FEBS Lett 402, 1-3 [PubMed]
  3. Bakos É, Evers R, Szakács G, Tusnády GE, Welker E, Szabó K, de Haas M, van Deemter L, Borst P, Váradi A and Sarkadi B (1998)
    Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain.
    J Biol Chem 273, 32167-75 [PubMed]
  4. Bakos É, Evers R, Calenda G, Tusnády GE, Szakács G, Váradi A and Sarkadi B (2000)
    Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1).
    J Cell Sci 113 (Pt 2), 4451-61 [PubMed]
  5. Kern A, Szentpétery Z, Liliom K, Bakos E, Sarkadi B and Váradi A (2004)
    Nucleotides and transported substrates modulate different steps of the ATPase catalytic cycle of MRP1 multidrug transporter.
    Biochem J 380, 549-60 [PubMed]
  6. Szentpétery Z, Kern A, Liliom K, Sarkadi B, Váradi A and Bakos E (2004)
    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.
    J Biol Chem 279, 41670-8 [PubMed]
  7. Deeley RG, Westlake C and Cole SP (2006)
    Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins.
    Physiol Rev 86, 849-99 [PubMed]
  8. Bodó A, Bakos É, Szeri F, Váradi A and Sarkadi B (2003)
    Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions.
    J Biol Chem 278, 23529-37 [PubMed]
  9. Bodó A, Bakos E, Szeri F, Váradi A and Sarkadi B (2003)
    The role of multidrug transporters in drug availability, metabolism and toxicity.
    Toxicol Lett 140-141, 133-43 [PubMed]
  10. Iliás A, Urbán Z, Seidl TL, Le Saux O, Sinkó E, Boyd CD, Sarkadi B and Váradi A (2002)
    Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).
    J Biol Chem 277, 16860-7 [PubMed]