R. Daoud et al., Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17, J BIOL CHEM, 276(15), 2001, pp. 12324-12330
MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown t
o confer resistance to structurally dissimilar drugs, Studies of MRP1 topol
ogy suggested the presence of a hydrophobic N-domain with five potential me
mbrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2
) by an intracellular linker domain (LO). MRP1-mediated multidrug resistanc
e is thought to be due to enhanced drug efflux. However, little is known ab
out MRP1-drug interaction and its drug binding site(s). We previously devel
oped several photoreactive probes to study MRP1-drug interactions. In this
report, we have used eight MRP1-HA variants that were modified to have hema
gglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exh
austive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA vari
ants revealed the same profile of photolabeled peptides as seen for wild ty
pe MRP1. Photolabeling of the different MRP1-HA variants followed by digest
ion with increasing concentrations of trypsin or Staphylococcus aureus V8 p
rotease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identif
ied two small photolabeled peptides (similar to6-7 kDa) from MRP1-HA(574) a
nd MRP1-HA(1222). Based on the location of the HA epitopes in the latter va
riants together with molecular masses of the two peptides, the photolabeled
amino acid residues were localized to MRP1 sequences encoding transmembran
es 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MS
D2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were al
so photolabeled with a structurally different photoreactive drug, IACI, con
firming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug b
inding. Taken together, the results in this study provide the first delinea
tion of the drug binding site(s) of MRP1. Furthermore, our findings suggest
the presence of common drug binding site(s) for structurally dissimilar dr
ugs.