title: 6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.
abstract: Multiple drug resistance protein 4 (MRP4, ABCC4) belongs to the C subfamily of the ATP-binding cassette (ABC) transporter superfamily and participates in the transport of diverse antiviral and chemotherapeutic agents such as 6-mercaptopurine (6-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA). We have undertaken a comprehensive functional characterization of protein variants of MRP4 found in Caucasians and other ethnicities. A total of 11 MRP4 missense genetic variants (nonsynonymous SNPs), fused to green fluorescent protein (GFP), were examined in Xenopus laevis oocytes for their effect on expression, localization, and function of the transporter. Radiolabeled 6-MP and PMEA were chosen as transport substrates. All MRP4 protein variants were found to be expressed predominantly in the oocyte membrane. A total of four variants (Y556C, E757 K, V776I, and T1142 M) exhibited a 20% to 40% reduced expression level compared to the wild type. Efflux studies showed that 6-MP is transported by MRP4 in unmodified form. Compared to wild-type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6-MP transport, while the amino acid exchange Y556C in the Walker(B) motif displayed significantly higher transport of PMEA. The transport properties of the other variants were comparable to wild-type MRP4. Our study shows that Xenopus oocytes are well suited to characterize MRP4 and its protein variants. Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates.
authors.affiliation: Institute of Pharmacology, University of Mainz, Mainz, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Janke
authors.forename: Sherif
authors.initials: S
authors.lastname: Mehralivand
authors.forename: Dennis
authors.initials: D
authors.lastname: Strand
authors.forename: Ute
authors.initials: U
authors.lastname: Gödtel-Armbrust
authors.forename: Alice
authors.initials: A
authors.lastname: Habermeier
authors.forename: Ulrike
authors.initials: U
authors.lastname: Gradhand
authors.forename: Christine
authors.initials: C
authors.lastname: Fischer
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Toliat
authors.forename: Peter
authors.initials: P
authors.lastname: Fritz
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
authors.forename: Matthias
authors.initials: M
authors.lastname: Schwab
authors.forename: Martin F
authors.initials: MF
authors.lastname: Fromm
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.forename: Leszek
authors.initials: L
authors.lastname: Wojnowski
authors.forename: Ellen I
authors.initials: EI
authors.lastname: Closs
authors.forename: Thomas
authors.initials: T
authors.lastname: Lang
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015122:Mercaptopurine / Q000493:pharmacokinetics*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D063065:Organophosphonates / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014982:Xenopus laevis
chemical_list: D000225:Adenine; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D015122:Mercaptopurine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D063065:Organophosphonates; D011993:Recombinant Fusion Proteins; C073492:ABCC4 protein, human; C485184:enhanced green fluorescent protein; C053001:adefovir