Characterization of binding of leukotriene C-4 by human multidrug resistance protein - Evidence of differential interactions with NH2- and COOH-proximal halves of the protein

Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- o r S-methyl GSH-stimulated manner. How MRP1 binds and transports such struct urally diverse substrates is not known. We have used [H-3]leukotriene C-4 ( LTC4), a high affinity glutathione-conjugated physiological substrate, to p hotolabel intact MRP1, as well as fragments of the protein expressed in ins ect cells. These studies revealed that: (i) LTC4 labels sites in the NH2- a nd COOH-proximal halves of MRP1, (ii) labeling of the NH2-half of MRP1 is l ocalized to a region encompassing membrane-spanning domain (MSD) 2 and nucl eotide binding domain (NBD) 1, (iii) labeling of this region is dependent o n the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 an d MSD2, but not on the presence of MSD1, (iv) labeling of the NH2-proximal site is preferentially inhibited by S-methyl GSH, (v) labeling of the COON- proximal half of the protein occurs in a region encompassing transmembrane helices 14-17 and appears not to require NBD2 or the cytoplasmic COOH-termi nal region of the protein, (vi) labeling of intact MRP1 by LTC4 is strongly attenuated in the presence of ATP and vanadate, and this decrease in label ing is attributable to a marked reduction in LTC4 binding to the NH2-proxim al site, and (vii) the attenuation of LTC4 binding to the NH2-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at N BD2. These data suggest that MRP1-mediated transport involves a conformatio nal change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC4 binds to one of two sites composed, at least in part, of elemen ts in the NH2-proximal half of the protein.