6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.
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.
Janke
Daniel
D
Institute of Pharmacology, University of Mainz, Mainz, Germany.
Mehralivand
Sherif
S
Strand
Dennis
D
Gödtel-Armbrust
Ute
U
Habermeier
Alice
A
Gradhand
Ulrike
U
Fischer
Christine
C
Toliat
Mohammad R
MR
Fritz
Peter
P
Zanger
Ulrich M
UM
Schwab
Matthias
M
Fromm
Martin F
MF
Nürnberg
Peter
P
Wojnowski
Leszek
L
Closs
Ellen I
EI
Lang
Thomas
T
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
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