Arsenic Triglutathione [As(GS)3] Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) Is Selectively Modified by Phosphorylation of Tyr920/Ser921 and Glycosylation of Asn19/Asn23

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)(3)]. In this study, marked differences in As(GS)(3) transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) (K-m 3.8 mu M and V-max 307 pmol/mg per minute) and HeLa (K-m 0.32 mu M and V-max 42 pmol/mg per minute) cells. Mutant MRP1 lacking N-linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low K-m and V-max values for As(GS)(3), similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, bothWT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)(3) (3-6 mu M), regardless of the cell line. Kinetic parameters of As(GS) 3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)(3) like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)(3) transport. The kinetics of As(GS)(3) transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)(3) transporter, allowing arsenic detoxification over a broad concentration range.