Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1).
The 190-kDa ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) encoded by the MRP1/ABCC1 gene mediates the active cellular efflux of glucuronide, glutathione and sulfate conjugates. It can also confer resistance to a diverse spectrum of chemotherapeutic agents and transport a variety of toxicants. In the present study, we examined 10 MRP1/ABCC1 missense genetic variants [non-synonymous single nucleotide polymorphisms (SNPs)] to determine whether or not they affect expression or function of the transporter. Variants 218C>T (Thr73Ile), 257C>T (Ser92Phe), 350C>T (Thr117Met), 689G>A (Arg230Gln), 1898G>A (Arg633Gln), 2168G>A (Arg723Gln), 2965G>A (Ala989Thr), 3140G>C (Cys1047Ser), 3173G>A (Arg1058Gln) and 4535C>T (Ser1512Leu) were recreated using site-directed mutagenesis and transfected into human embryonic kidney cells. Immunoblotting experiments showed that all mutant proteins were expressed at levels comparable to wild-type MRP1. Vesicular transport assays revealed that the Ala989Thr mutation caused a significant decrease in estradiol 17beta-glucuronide transport due to a decrease in apparent affinity (Km) for this organic anion. The transport properties of the other mutants were comparable to wild-type MRP1. When the MRP1/ABCC1 non-synonymous SNPs were evaluated by the SIFT algorithm using subsets of homologs and orthologs of MRP1/ABCC1, Arg230Gln, Val353Met, Arg433Ser, Gly671Val and Arg1058 mutations were predicted to be deleterious, whereas the PolyPhen algorithm predicted Ser92Phe and Gly671Val to be potentially damaging. Thus most predictions of these algorithms were not in accordance with our experimental results. In conclusion, our data suggest that none of the MRP1/ABCC1 variants studied are likely by themselves to have major deleterious effects in healthy individuals, and the SIFT and PolyPhen algorithms appear to be poor predictors of the phenotypic consequences of these MRP1 mutations at least in vitro.
Létourneau
Isabelle J
IJ
Department of Pharmacology and Toxicology, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada.
Deeley
Roger G
RG
Cole
Susan P C
SP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000483:Alleles; D000595:Amino Acid Sequence; D000838:Anions; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D017124:Conserved Sequence; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D005822:Genetic Vectors; D020719:Glucuronides / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D014162:Transfection
D000255:Adenosine Triphosphate; D000838:Anions; D000970:Antineoplastic Agents; D004958:Estradiol; D020719:Glucuronides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C421109:multidrug resistance-associated protein 1