The human multidrug resistance protein 4 (MRP4, ABCC4): functional analysis of a highly polymorphic gene

Abstract

ABCC4 encodes multidrug resistance protein 4 (MRP4), a member of the ATP-binding cassette family of membrane transporters involved in the efflux of endogenous and xenobiotic molecules. The aims of this study were to identify single nucleotide polymorphisms of ABCC4 and to functionally characterize selected nonsynonymous variants. Resequencing was performed in a large ethnically diverse population. Ten nonsynonymous variants were selected for analysis of transport function based on allele frequencies and evolutionary conservation. The reference and variant MRP4 cDNAs were constructed by site-directed mutagenesis and transiently transfected into human embryonic kidney cells (HEK 293T). The function of MRP4 variants was compared by measuring the intracellular accumulation of two antiviral agents, azidothymidine (AZT) and adefovir (PMEA). A total of 98 variants were identified in the coding and flanking intronic regions of ABCC4. Of these, 43 variants are in the coding region, and 22 are nonsynonymous. In a functional screen of ten variants, there was no evidence for a complete loss of function allele. However, two variants (G187W and G487E) showed a significantly reduced function compared to reference with both substrates, as evidenced by higher intracellular accumulation of AZT and PMEA compared to the reference MRP4 (43 and 69% increase in accumulation for G187W compared with the reference MRP4, with AZT and PMEA, respectively). The G187W variant also showed decreased expression following transient transfection of HEK 293T cells. Further studies are required to assess the clinical significance of this altered function and expression and to evaluate substrate specificity of this functional change.

Figure 1

Transmembrane prediction for MRP4 and localization of the non-synonymous variants and non-functional mutant constructed for this study (NBDs: nucleotide binding domains, EC: evolutionarily conserved, EU: evolutionarily unconserved, red circles: non-synonymous SNPs, green circles: synonymous SNPs, black circle: non-functional mutant)

Figure 2

Population genetics statistics for ABCC4. Shown are the π (A) and θ (B) parameters for ABCC4 in the different populations studied (mean ± SD). Values were calculated for the total (solid), coding (diagonal), non-coding (open), synonymous (checkered), and non-synonymous (gray) variant sites.

Figure 3

Sequence alignments and frequencies of ABCC4 haplotypes. For simplification, only variants in the coding and UTR regions were used for haplotype estimation. A total of 71 haplotypes were constructed using the PHASE method with the sites present in more than 5% in at least one population. Only the 45 haplotypes that were found more than once are shown. Haplotypes have been aligned according to sequence similarity. The color code is as follows: a black box means reference sequence; red and green boxes correspond to non-synonymous and synonymous changes, respectively; the variants in the 5’ and 3’ UTR regions are colored in yellow. Haplotypes were numbered according to evolutionary considerations and population frequency (*2 being the most common), but *1, which has no variant sites, has been set as the reference.

Figure 4

Intracellular accumulation of AZT (gray bars) and PMEA (open bars) for reference and variant MRP4. Accumulation is expressed relative to reference MRP4 for negative controls (A) and the selected variants (B). Cells were incubated at 37°C with 100 nM AZT for 30 min, or 100 nM PMEA for one h. The values shown are the mean ± standard deviation, for n = 6−12 replicates. Significant differences between reference and variants were assessed by a Student's t-test with Bonferroni correction for multiple testing (* p < 0.005, ** p < 0.001)

Figure 5

Concentration-dependence of (A) AZT and (B) PMEA accumulation in cells expressing either the reference MRP4 or one of the variants. The values represent the mean ± SD of n=3 (AZT) or n=4 (PMEA) determinations (■ reference, ◆ P78A, ▲ G187W, △ G487E, and ○ C956S).

Figure 6

Expression of MRP4 in transfected HEK 293T cells. Protein was isolated 24 hours after transfection with pcDNA5/FRT, MRP4 reference and MRP4 variants and immunoreactive MRP4 was detected by Western blot. GAPDH (lower panel) was measured as a loading control.

Figure 7

Localization of MRP4 proteins by immunocytochemistry. Cells were grown on cover slips and transfected with the different variants. Twenty four h after transfection, cells were incubated with a monoclonal antibody to MRP4 (M4-I10) and a fluorescent goat anti-rat secondary antibody. A: Empty vector, B: MRP4 reference, C: P78A, D: G187W, E: P403L, F: G487E, G: C956S, H: G538D. All images are shown at 200× magnification.