Studies of the Misprocessing Mutations R1202D and E1204K in the Drug and Organic Anion Transporter, MRP1 (ABCC1) in Cultured HEK cells

Abstract

Multidrug resistance protein 1 (MRP1) is a drug and organic anion transporter of the ATP-binding cassette superfamily. Previous studies showed that opposite charge substitutions of Arg1202 or Glu1204 in transmembrane helix (TM) 16 cause a >80% reduction in MRP1 levels when expressed in human embryonic kidney (HEK) cells. These substitutions disrupt the folding and/or assembly of MRP1 which targets it for degradation. Attempts were made to enhance levels of the R1202D and E1204K misprocessing mutants by incubating transfected HEK cells at 30 ºC or 27 ºC. At both temperatures, cells expressed both fully glycosylated and underglycosylated mutants at levels 60–70% lower than wild-type MRP1in cells grown at 37 ºC. The subcellular localization patterns of the two mutants were similar to wild-type MRP1 at all three temperatures, with most of the transporter at the plasma membrane at 37 ºC, and in the endoplasmic reticulum at 30 ºC or 27 ºC. Thus, although poorly expressed, the R1202D and E1204K mutants retained the ability to traffic to the plasma membrane. Attempts were also made to enhance R1202D and E1204K levels by exposing transfected HEK cells to chemical chaperones. Dimethyl sulfoxide and glycerol increased E1204K levels by 20-30% but decreased or had no effect on R1202D and wild-type MRP1. 4-Phenylbutyric acid had little or no effect on either wild-type or mutant MRP1. Thus both mutants were relatively resistant to rescue by chemical chaperones. Finally, a “second-site rescue mutation” approach was taken, guided by an atomic homology model of MRP1. Mutations of Tyr1133 alone decreased MRP1 levels, like R1202D; however, although substituting TM15-Tyr1133 with Phe, His and Ala in R1202D was predicted to re-establish TM15-TM16 bonding interactions, levels of this mutant did not increase. E1204K levels were also not improved by substituting TM17-Val1248 with Asp or Glu although these substitutions were predicted to re-establish TM16-TM17 bonds disrupted in E1204K. These results suggest that the bonding interactions of Arg1202 and Glu1204 with other amino acids predicted by the MRP1 homology model used in this study are insufficient to predict the critical helix-helix interactions necessary for stable MRP1 expression in mammalian cells.