Mutation of Trp(1254) in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity andresults in loss of methotrexate transport activity
K. Ito et al., Mutation of Trp(1254) in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity andresults in loss of methotrexate transport activity, J BIOL CHEM, 276(41), 2001, pp. 38108-38114
The ATP-binding cassette (ABC) proteins comprise a large superfamily of tra
nsmembrane transporters that utilize the energy of ATP hydrolysis to transl
ocate their substrates across biological membranes. Multidrug resistance pr
otein (AMP) 2 (AJBCC2) belongs to subfamily C of the ABC superfamily and, w
hen overexpressed in tumor cells, confers resistance to a wide variety of a
nticancer chemotherapeutic agents. MRP2 is also an active transporter of or
ganic anions such as methotrexate (MTX), estradiol glucuronide (E(2)17 beta
G), and leukotriene C-4 and is located on the apical membrane of polarized
cells including hepatocytes where it acts as a biliary transporter. We rece
ntly identified a highly conserved tryptophan residue in the related MRP1 t
hat is critical for the substrate specificity of this protein. In the prese
nt study, we have examined the effect of replacing the analogous tryptophan
residue at position 1254 of MRP2. We found that only nonconservative subst
itutions (Ala and Cys) of Trp(1254) eliminated [H-3]E(2)17 betaG transport
by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effec
t. In addition, only the most conservatively substituted mutant (W1254Y) tr
ansported [H-3]leukotriene C-4, whereas all other substitutions eliminated
transport of this substrate. On the other hand, all substitutions of Trp 12
54 eliminated transport of [H-3]MTX. Finally, we found that sulfinpyrazone
stimulated [H-3]E(2)17 betaG transport by wild-type MRP2 4-fold, whereas tr
ansport by the Trp(1254) substituted mutants was enhanced 6-10-fold. In con
trast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wildt
ype MRP2 or the MRP2-Trp(1254) mutants. Taken together, our results demonst
rate that Trp(1254) plays an important role in the ability of MRP2 to trans
port conjugated organic anions and identify this amino acid in the putative
last transmembrane segment (TM17) of this ABC protein as being critical fo
r transport of MTX.