Monoclonal antibodies that inhibit the transport function of the 190-kDa multidrug resistance protein, MRP. Localization of their epitopes to the nucleotide-binding domains of the protein.
Multidrug resistance in tumor cells is often accompanied by overexpression of multidrug resistance protein (MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding cassette superfamily of transport proteins. MRP mediates ATP-dependent transport of a variety of conjugated organic anions and can also transport several unmodified xenobiotics in a glutathione-dependent manner. To facilitate structure-function studies of MRP, we have generated a panel of MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2, -3, -4, and -6, bind intracellular conformation-dependent epitopes, and we have shown that they can inhibit the transport of several MRP substrates. Binding competition and immunoprecipitation assays indicated that mAbs QCRL-4 and -6 probably recognize the same detergent-sensitive epitope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the mAbs with MRP. Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 and -3 are amino acids 617-858 and 617-932, respectively, which encompass the NH2-proximal nucleotide-binding domain (NBD). In contrast, the epitope bound by mAb QCRL-4 localized to amino acids 1294-1531, a region that contains the COOH-proximal NBD. However, none of the mAbs inhibited photolabeling of intact MRP with 8-azido-[alpha-32P]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering with substrate binding or by trapping MRP in a conformation that does not allow transport to occur. Our results also demonstrate for the first time that the NBDs of MRP can be expressed as soluble polypeptides that retain a native conformation.
Hipfner
D R
DR
Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Mao
Q
Q
Qiu
W
W
Leslie
E M
EM
Gao
M
M
Deeley
R G
RG
Cole
S P
SP
D018528:ATP-Binding Cassette Transporters / Q000276:immunology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001386:Azides; D001667:Binding, Competitive; D001692:Biological Transport; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000494:pharmacology; D017997:Leukotriene C4 / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments / Q000276:immunology; D011233:Precipitin Tests; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D001386:Azides; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate