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

Citation
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
Citations number
73
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
41
Year of publication
2001
Pages
38108 - 38114
Database
ISI
SICI code
0021-9258(20011012)276:41<38108:MOTITM>2.0.ZU;2-B
Abstract
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.