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-dep endent manner. To facilitate structure-function studies of MRP, we have gen erated a panel of MRP-specific monoclonal antibodies (mAbs), Four of these mAbs, QCRL-2, -3, -4, and -6, bind intracellular conformation-dependent epi topes, and we have shown that they can inhibit the transport of several MRP substrates, Binding competition and immunoprecipitation assays indicated t hat mAbs QCRL-4 and -6 probably recognize the same detergent-sensitive epit ope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlap ping epitopes, Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the m Abs with MRP, Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 a nd -3 are amino acids 617-858 and 617-932, respectively, which encompass th e NH2-proximal nucleotide-binding domain (NBD), In contrast, the epitope bo und by mAb QCRL-4 localized to amino acids 1294-1531, a region that contain s the COOH-proximal NBD. However, none of the mAbs inhibited photo-labeling of intact MRP with 8-azido-[alpha-P-32]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering w ith substrate binding or by trapping MRP in a conformation that does not al low transport to occur. Our results also demonstrate for the first time tha t the NBDs of MRP can be expressed as soluble polypeptides that retain a na tive conformation.