Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP)

Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17␤-estradiol 17-(␤-D-glucuronide) (E 2 17␤G), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu 1089 of MRP1 as being critical for anthracycline resistance. However, Glu 1089 mutations had no effect on E 2 17␤G transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala 1239 in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E 2 17␤G transport 3-fold. Any mutation of mrp1 Ala 1239 , including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr 1242 for E 2 17␤G transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E 2 17␤G transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C 4 transport ϳ2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/ T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C 4 and E 2 17␤G. These results demonstrate the important role of Thr 1242 for E 2 17␤G transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.Human multidrug resistance protein 1 (MRP1), 1 a 190-kDa glycoprotein, is a member of the ATP-binding cassette (ABC) transporter superfamily (1-3). The predicted structure of MRP1 differs from that of a typical eukaryotic ABC transporter such as P-glycoprotein (P-gp). It contains a P-gp-like core region, which consists of two membrane-spanning domains (MSDs), each containing six transmembrane (TM) ␣-helices, and two nucleotide binding domains. However, MRP1 contains a third MSD predicted to consist of five TM ␣-helices with an extracellular NH 2 terminus and a cytoplasmic linker connecting the additional MSD with the core region (3-7). Like P-gp, MRP1 confers resistance to many commonly used, structurally diverse natural product chemotherapeutic agents including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (7-10). However, several lines of evidence suggest that MRP1 and P-gp confer resistance to these drugs by different mechanisms. In vitro studies using P-gp-enriched membrane vesicles or purifi...

Section: Methodsmentioning

confidence: 99%

Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity

Zhang¹,

Cole²,

Deeley

2001

“…Fulllength wild-type and COOH-terminal truncated, mutated, and hybrid membrane proteins were subjected to SDS-PAGE (7.5%), whereas dual half-expressed proteins were resolved using SDS-PAGE gradient gels (7.5-15%). Immunoblots of intact proteins were probed with mAb MRPr1 (35,36), whereas the dual expressed NH 2 -half was immunodetected using mAb MRPr1, and the COOH-half was immunodetected with mAb MRPm5 (37). Antibody-protein interactions were detected using horseradish peroxidase secondary antibodies utilizing an enhanced chemiluminescence, and the relative expression of MRP1 fragments was determined by densitometry.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Functionally Important Elements in the Multidrug Resistance Protein 1 COOH-terminal Region

Westlake

Payen

Gao

et al. 2004

The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOHterminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

“…Following transfer of the membrane proteins to Immobilon-P membranes (Millipore), MRP1 polypeptides were detected using an enhanced chemiluminescence kit (PerkinElmer Life Sciences) and monoclonal antibodies QCRL-1 and MRPm6 for MRP1 and MRPr1, which recognize a common epitope in MRP1 and mrp1 (46,47).…”

Section: Methodsmentioning

confidence: 99%

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

Qian

Grant

Westlake

et al. 2002

We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH 2 -and COOH-proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.