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) (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, both WT- 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 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.
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Caley B
CB
Shukalek
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Diane P
DP
Swanlund
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Rodney K
RK
Rousseau
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Kevin E
KE
Weigl
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Vanessa
V
Marensi
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada.
Susan P C
SP
Cole
Department of Physiology (C.B.S., D.P.S., R.K.R., V.M., E.M.L.) and Membrane Protein Disease Research Group (C.B.S., D.P.S., R.K.R., V.M., E.M.L.), University of Alberta, Edmonton, Alberta, Canada. Department of Pathology and Molecular Medicine and Division of Cancer Biology and Genetics (K.E.W., S.P.C.C.), Queen's University, Kingston, Ontario, Canada eleslie@ualberta.ca.
Elaine M
EM
Leslie
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001151:Arsenic / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism; D008970:Molecular Weight; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001151:Arsenic; D004791:Enzyme Inhibitors; D004958:Estradiol; D005965:Glucuronates; D008727:Methotrexate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D014357:Trypsin; C409215:17-glucuronosylestradiol; C421109:multidrug resistance-associated protein 1