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

Abstract

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) (Km 3.8 μM and Vmax 307 pmol/mg per minute) and HeLa (Km 0.32 μM and Vmax 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 Km and Vmax 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 μM), regardless of the cell line. Kinetic parameters of As(GS)3 for HEKAsn19/ 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-WTMRP1, 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.