A Positively Charged Amino Acid Proximal to the C-terminus of TM17 of MRP1 Is Indispensable for GSH-dependent Binding of Substrates and for Transport of LTC4

Biochemistry. 2002 Dec 3;41(48):14132-40. doi: 10.1021/bi026443s.

Abstract

MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / genetics
  • Animals
  • Arginine / chemistry*
  • Arginine / genetics
  • Arginine / metabolism
  • Binding Sites
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Drug Resistance, Multiple* / genetics
  • Glutathione / chemistry*
  • Glutathione / metabolism
  • Humans
  • LLC-PK1 Cells
  • Leukotriene C4 / chemistry*
  • Leukotriene C4 / metabolism
  • Methionine / genetics
  • Multidrug Resistance-Associated Proteins / biosynthesis
  • Multidrug Resistance-Associated Proteins / chemistry*
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / metabolism
  • Mutagenesis, Site-Directed / genetics
  • Peptide Fragments / biosynthesis
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Photoaffinity Labels / metabolism
  • Protein Transport
  • Spodoptera / genetics
  • Sterols / metabolism
  • Swine

Substances

  • Multidrug Resistance-Associated Proteins
  • Peptide Fragments
  • Photoaffinity Labels
  • Sterols
  • agosterol A
  • Leukotriene C4
  • Arginine
  • Methionine
  • Glutathione
  • Alanine
  • multidrug resistance-associated protein 1