ABCMdb

PMID: 22290738

Arlanov R, Porter A, Strand D, Brough R, Karpova D, Kerb R, Wojnowski L, Schwab M, Lang T
Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells.
Hum Mutat. 2012 Apr;33(4):750-62. doi: 10.1002/humu.22041. Epub 2012 Feb 28., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). Login to comment 6 ABCC2 p.Arg1174His R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Login to comment 7 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Login to comment 8 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu Contrary to protein data, the double variant V1188E/ C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. Login to comment 10 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation. Login to comment 42 ABCC2 p.Cys1515Tyr ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Val1188Glu Materials and Methods Construction of Enhanced Green Fluorescent Protein (EGFP) Tagged and Untagged hABCC2(V1188E/C1515Y) Expression Plasmids A 5.3-kB cDNA encoding the human ABCC2 variant V1188E/C1515Y (GenBank accession number U49248.1) was subcloned into the vector pGEM3 provided by Professor Dr Piet Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands [Evers et al., 1998]. Login to comment 46 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu The resulting plasmid was ABCC2(V1188E/C1515Y)- EGFP.pEGFP-N1. Login to comment 48 ABCC2 p.Cys1515Tyr ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Val1188Glu The PCR fragment was cloned into the SpeI/NotI site of ABCC2(V1188E/C1515Y)-EGFP.pEGFP-N1 obtaining ABCC2(V1188E/C1515Y).pEGFP-N1 without an EGFP tag. Login to comment 169 ABCC2 p.Arg1150His ABCC2 p.Val1188Glu ABCC2 p.Ile1173Phe ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu ABCC2 p.Ile1036Thr ABCC2 p.Ile670Thr Computational comparative genomic studies using PolyPhen 2 predicted that nine of these ABCC2 variants (F39Y, D333G, I670T, I1036T, R1174H, R1181L, V1188E, N1244K, and P1291L) would be located within the transmembrane regions, close to the ATP-binding domain of ABCC2 or close to two missense variants (I1173F, R1150H) causing Dubin-Johnson syndrome [Keitel et al., 2003; Mor-Cohen et al., 2001]. Login to comment 170 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu Five of the variants (D333G, R1174H, R1181L, N1244K, and P1291L) were predicted to have a functional effect. Login to comment 173 ABCC2 p.Cys1515Tyr ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Val1188Glu Because V1188E and C1515Y are linked, only the double variant V1188E/C1515Y was investigated. Login to comment 174 ABCC2 p.Ile1173Phe The Dubin-Johnson syndrome- causing variant ABCC2 I1173F served as a positive control for reduced ABCC2 expression. Login to comment 176 ABCC2 p.Arg353His ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu ABCC2 p.Ile1036Thr ABCC2 p.Ile670Thr ABCC2 p.Gly921Ser ABCC2 p.Thr486Ile Immunoblots of the variants F39Y, R353H, T486I, I670T, G921S, I1036T, N1244K, and P1291L showed similar expression compared with WT (range 85-110% of WT). Login to comment 177 ABCC2 p.Ile1173Phe ABCC2 p.Arg1174His ABCC2 p.Asp333Gly Staining of ABCC2 was weaker for D333G, I1173F, R1174H, and Table 1. Login to comment 178 ABCC2 p.Arg353His ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu ABCC2 p.Ile1036Thr ABCC2 p.Ile670Thr ABCC2 p.Gly921Ser ABCC2 p.Thr486Ile ABCC2 Missense Variants Selected for Study on Expression, Localization, and Function rs# NCBI Genetic variationa Amino acid Ethnicity Allele frequency (%) Nb Data source Predicted phenotypec rs927344 c.116T>A F39Y AA 2.3 86 Current study Benign AA 2 200 pharmGKB KO 0 94 Current study rs17222674 c.998A>G D333G AA 0 88 Current study Probably damaging AA 1 200 pharmGKB KO 0 94 Current study rs7080681 c.1058G>A R353H AA 6.7 90 Current study Benign AA 3.5 200 pharmGKB KO 0 94 Current study rs17222589 c.1457C>T T486I AA 0 84 Current study Benign KO 3.7 82 Current study JA 5 20 pharmGKB JA 2.3 144 Itoda et al. (2002) rs17222632 c.2009T>C I670T AA 1.1 92 Current study Benign AA 1.5 200 pharmGKB KO 0 92 Current study rs41318029 c.2761G>A G921S AA 0 84 Current study Benign KO 0 92 Current study CA 1 120 NCBI dbSNP rs45441199 c.3107T>C I1036T AA 0 96 Current study Benign AA 0.5 200 pharmGKB KO 0 94 Current study CA 0.5 198 pharmGKB CA 1 120 NCBI dbSNP rs139188247 c.3521G>A R1174H AA 2.3 86 Current study Probably damaging KO 0 94 Current study JA 1 144 Itoda et al. (2002) rs8187692 c.3542G>T R1181L AA 5.8 86 Current study Probably damaging AA 8.5 200 pharmGKB KO 0 96 Current study rs17222723 c.3563T>A V1188E AA 5.8 86 Current study Benign AA 6.5 200 pharmGKB KO 0 96 Current study CA 7.5 200 pharmGKB c.3732T>G N1244K JA 1.5 144 Itoda et al. (2002) Possibly damaging rs17216317 c.3872C>T P1291L AA 4.7 86 Current study Probably damaging AA 2 86 pharmGKB KO 0 86 Current study CA 0.5 196 pharmGKB rs8187710 c.4544G>A C1515Y AA 13 92 Current study Benign AA 19.6 194 pharmGKB KO 0 94 Current study CA 8 198 pharmGKB AA, African-American; CA, Caucasian; KO, Korean; JA, Japanese. Login to comment 182 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1181Leu R1181L (range 30-55% of WT, P < 0.01), whereas higher expression was found for the double variant V1188E/C1515Y (150% of WT, P < 0.01) (Supp. Fig. S5). Login to comment 183 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu Cellular Localization of ABCC2 WT and Variants in HEK 293 Cells The variable ABCC2 expression in HEK 293 cells carrying the D333G, R1174H, R1181L, and V1188E/C1515Y variants suggests that processing or protein stability of ABCC2 could be affected by these variants. Login to comment 186 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly For all variants except D333G and R1174H, ABCC2 protein was localized to intracellular membranous structures, indicating that processing and targeting of these variants seems to be comparable to WT. Login to comment 187 ABCC2 p.Asp333Gly In contrast, the transmembrane variant D333G showed a punctuated localization in the plasma membrane. Login to comment 188 ABCC2 p.Arg1174His Moreover, sparse intracellular distribution of the variant R1174H was found by examination of several fields in separate transfection experiments. Login to comment 204 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His Interestingly, the variable noninhibited EGFP value measured by the peak channel was approximately 800-fold for ABCC2 (WT) and approximately 500-fold (R1174H) and 1500- fold (V1188E/C1515Y) for ABCC2 variants compared with nonfluorescing cells (HT1080). Login to comment 205 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu With the exception of V1188E/C1515Y, the degree of downregulation after 72 hr did not seem to depend upon the type of variant construct used and the particular clone analyzed. Login to comment 206 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu The EGFP value was approximately 10-fold for ABCC2 (WT and 11 variants) compared with 20-fold for the ABCC2 double variant V1188E/C1515Y. Login to comment 210 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu D333G, R1174H, and R1181L exhibited a 40%, 70%, and 35% reduction in protein expression, respectively, whereas the double variant V1188E/C1515Y resulted in a 150% increase in protein expression compared with WT (P < 0.01). Login to comment 214 ABCC2 p.Asn1244Lys The N1244K variant showed a significant decrease of transport activity, with a 15% and 26% efflux of GS-MF and GS-MCB, respectively, compared with ABCC2 WT (100%) (P < 0.01, one-way ANOVA followed by Dunnett`s post test). Login to comment 215 ABCC2 p.Arg1174His The activity of R1174H was reduced by approximately 50% for both substrates compared with WT (GS-MF, 56%; GS-MCB, 54%; P < 0.01). Login to comment 216 ABCC2 p.Arg1181Leu Interestingly, the variant R1181L resulted in an increased effluxofGS-MF(137%,P< 0.01)andadecreasedeffluxofGS-MCB compared with WT (59%, P < 0.01). Login to comment 217 ABCC2 p.Arg353His ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Asp333Gly ABCC2 p.Pro1291Leu ABCC2 p.Ile1036Thr ABCC2 p.Gly921Ser ABCC2 p.Thr486Ile The D333G, R353H, T486I, G921S, and P1291L variants showed a lower transport activity for GS-MCB (71%, 60%, 75%, 63%, and 51% of WT, P < 0.01) but not for GS-MF, whereas the variants F39Y, I1036T, and V1188E/C1515Y did not alter ABCC2-mediated transport for either substrate. Login to comment 219 ABCC2 p.Asn1244Lys Only N1244K caused a major decrease of ABCC2-specific activity (up to 80% for GS-MF and GS-MCB when compared with WT), whereas protein expression was similar to WT. Login to comment 220 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu In contrast, R1174H (30% of WT) and R1181L (65% of WT) caused a twofold increase in specific GS-MF and/or GS-MCB efflux Figure 5. Login to comment 230 ABCC2 p.Pro1291Leu The P1291L variant caused a 50% decrease in specific GS-MCB transport activity without alteration of ABCC2 protein. Login to comment 231 ABCC2 p.Cys1515Tyr Notably, the double variant V11888E/C1515Y showed the highestproteinexpressionlevelbutcauseda40%decreaseinspecific efflux of GS-MCB and GS-MF. Login to comment 233 ABCC2 p.Arg1174His Consistent with the findings in HEK 293 cells, ABCC2 WT and all variants except R1174H were predominantly localized to the cell surface. Login to comment 234 ABCC2 p.Arg1174His The R1174H variant was localized primarily in the cytoplasm with an endoplasmic reticulum (ER)-like pattern distribution. Login to comment 235 ABCC2 p.Asp333Gly The D333G variant, which showed punctuated localization in HEK 293 cells, was predominantly localized to the Rht14-10 cell surface. Login to comment 236 ABCC2 p.Cys1515Tyr ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Arg1181Leu EGFP Kinetics of the ABCC2 Variants D333G, R1174H, R1181L, and V1188E/C1515Y To elucidate whether protein stability will affect degradation of ABCC2 after adding of doxycycline (1 μg/ml), we investigated EGFP kinetics of the ABCC2 D333G, R1174H, R1181L, and V1188E/C1515Y variants expressed by Rht14-10 cells. Login to comment 238 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu No differences in EGFP kinetics for the ABCC2 variants D333G, R1174H, R1181L, and V1188E/C1515Y were detected, indicating no changes in protein stability (Supp. Fig. S11). Login to comment 240 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly Subcellular localization of ABCC2 variants D333G and R1174H in Rht14-10 cells. Login to comment 252 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu Compared with the ABCC2 reference sequence, the expression of ABCC2wasreducedforthethreemissensevariantsD333G,R1174H, and R1181L (range 30-65% of WT), whereas the double variant V1188E/C1515Y revealed a significantly higher expression (150% of WT). Login to comment 253 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu Altered protein expression of the ABCC2 variants D333G, R1174H, R1181L, and V1188E/C1515Y seems not to be the direct consequence of impaired or increased protein stability (Supp. Fig. S11), thereby suggesting alteration in synthesis or stability of the mutant ABCC2 mRNA. Login to comment 255 ABCC2 p.Arg1174His Regarding the localization of ABCC2 in the HT1080 cells, ABCC2-EGFP could be detected in the plasma membrane by confocal fluorescence microscopic analysis for each of the ABCC2 variants except R1174H. Login to comment 258 ABCC2 p.Asp333Gly Moreover, D333G plasma membrane localization was reduced in HEK 293 cells, and showed a striking punctiform pattern. Login to comment 260 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly Because the cell type may influence localization of ABCC2 and polarized cells may be a more physiological system, it remains still open whether the alteration in membrane localization of the two ABCC2 variants D333G and R1174H in HEK 293 and/or HT 1080 cells is due to missing proteins, which are required for sorting or a result of an incomplete polarity of the HEK 293 and HT1080 cells in culture. Login to comment 262 ABCC2 p.Asn1244Lys N1244K was associated with a maximal decrease of approximately 80% in overall efflux of GS-MF/MCB compared with WT, in contrast to the protein data. Login to comment 263 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu ABCC2 p.Pro1291Leu Moreover, the R1174H variant resulted in a decrease of overall transport activity of approximately 50% for both the substrates, GS-MF and GS-MCB, whereas only GS-MCB efflux was reduced with R1181L and P1291L. Login to comment 264 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu In the case of R1174H and R1181L, altered transport capacity was associated with a lower protein expression. Login to comment 265 ABCC2 p.Arg1181Leu As it is well known that transport activities may differ among various substrates when using the same variant, it is not surprising that the R1181L variant resulted in a twofold increase in efflux for GS-MF but not for GS-MCB. Login to comment 266 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu Interestingly, although the double variant V1188E/C1515Y showed the greatest expression of ABCC2 protein (150% of WT), this did not result in an increased specific efflux of GS-MF and GS-MCB because transport activities were virtually similar to WT. Login to comment 267 ABCC2 p.Arg353His ABCC2 p.Asp333Gly ABCC2 p.Ile1036Thr ABCC2 p.Ile670Thr ABCC2 p.Gly921Ser ABCC2 p.Thr486Ile Finally, GS-MF and/or GS-MCB efflux for all other ABCC2 variants (F39Y, D333G, R353H, T486I, I670T, G921S, and I1036T) showed only minor alterations compared with WT based on our definition of at least 50% change in transport activity. Login to comment 272 ABCC2 p.Arg1150His ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Ile1173Phe ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu The selected variants R1174H, R1181L, and V1188E (linked to C1515Y) from the present study, in addition to two recently identified variants that cause Dubin-Johnson syndrome (R1150H, I1173F), are located at the same gene region between the transmembrane helices TM15 and TM16. Login to comment 275 ABCC2 p.Asp333Gly ABCC2 p.Asn1244Lys ABCC2 p.Ile1036Thr Three of the four transmembrane variants studied (F39Y [TM1]), D333G [TM6]), I1036T [TM13], and N1244K [TM17]) were localized in the TM6 to TM17. Login to comment 276 ABCC2 p.Asp333Gly ABCC2 p.Asn1244Lys The impact of TM6 and TM17 on the transport function of ABCC2 is consistent with our observation for D333G (TM6) and N1244K (TM17), showing a decreased transport activity and/or expression. Login to comment 277 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala Moreover, a substitution of W1254 by conserved and nonconserved amino acids (e.g., W1254A, W1254Y) in TM17 seems to be critical for substrate binding and transport activity [Ito et al., 2001b]. Login to comment 278 ABCC2 p.Ile1036Thr In contrast, TM13 appears to be of minor importance for ABCC2 function, which is supported by our data that the I1036T variant, located at TM13, did not induce any alteration in proteins or functionality [Ryu et al., 2000]. Login to comment 279 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Pro1291Leu The total allele frequency for all functionally relevant ABCC2 protein variants (D333G, R1174H, R1181L, V1188E/C1515Y, and P1291L) was highest in the African-American subjects, at approximately 20%. Login to comment 280 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Pro1291Leu As shown by the haplotype analysis of our sequencing data, the functional variants D333G, R1174H, R1181L, and P1291L were not linked to each other or to other variants in African-Americans. Login to comment 281 ABCC2 p.Cys1515Tyr ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Val1188Glu The double variant V1188E/C1515Y has been associated with differences in tissue expression by several in vivo studies, resulting in possible consequences for disease susceptibility and/or drug response, underscoring the clinical importance of ABCC2 V1188E/C1515Y [Elens et al., 2009; Grisk et al., 2009; Meier et al., 2005; Ni et al., 2010; Sookoian et al., 2009; Wojnowski et al., 2005]. Login to comment 282 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu ABCC2 p.Pro1291Leu Because the R1174H, R1181L, and P1291L variants impaired transport activity by approximately 50%, considerably alteration of the pharmacokinetics of ABCC2 substrates may be expected, resulting in potential clinical consequences such as adverse drug reactions. Login to comment 284 ABCC2 p.Asp333Gly ABCC2 p.Asn1244Lys In contrast, the D333G and N1244K variants, which also significantly impaired ABCC2 transport activity, are extremely rare. Login to comment 288 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Pro1291Leu The human ABCC2 WT residue was highly conserved only for D333G, R1174H, R1181L, and P1291L compared with sequences of ABCC2 orthologs and other ABCC homologs. Login to comment 290 ABCC2 p.Asn1244Lys With the exception of N1244K, these data are in line with our in vitro results. Login to comment 291 ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu When PolyPhen 2 (http://genetics.bwh.harvard.edu/pph) was used to evaluate the ABCC2 missense variants in this study, D333G, R1174H, R1181L, N1244K, and P1291L were predicted as deleterious (Table 1). Login to comment 299 ABCC2 p.Arg1174His ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu Moreover, convincing evidence of highly variable GS-MF and GS-MCB transport activity and ABCC2 expression is provided by the functional characterization of ABCC2 nonsynony- mousvariants.TheD333G,R1174H,R1181L,N1244K,P1291L,and Table 2. Login to comment 300 ABCC2 p.Arg353His ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu ABCC2 p.Arg1174His ABCC2 p.Asp333Gly ABCC2 p.Arg1181Leu ABCC2 p.Asn1244Lys ABCC2 p.Pro1291Leu ABCC2 p.Ile1036Thr ABCC2 p.Ile670Thr ABCC2 p.Gly921Ser ABCC2 p.Thr486Ile Conservation of the ABCC2 Missense Variants Among Other ABCC Orthologs and Homologs Protein Speciesa F39Y D333G R353H T486I I670T G921S I1036T R1174H R1181L V1188E N1244K P1291L C1515Y ABCC2 Human F D R T I G I R R V N P C Mouse F D P K V S I R R K N P Y Rat F D S N V S I R R K N P Y Rabbit F D P N V G L R R I N P Y Rhesus F D R T M S I R R V N P Y ABCC1 Human Y D T T V G I R R L Y P Y Mouse Y D R T V G A R R L Y P C Rat Y D R T V V V R R L Y P C Macaque Y D T T V G I R R L Y P Y Dog Y D K T V G I R R L Y P C ABCC3 Human Y D P A V G F R D T Y E F Mouse Y N P T V V L R D T Y P F Rat Y D P T V G L R D A Y P F ABCC4 Human - E Y S V R L R R A Y P Y ABCC5 Human - Q A Y C P I H E E Y P E ABCC6 Human Y D P H V K I R P A A P S ABCC11 Human - C E K C T C H D R I Q F Multiple sequence alignment was performed using ClustalW (www.ebi.ac.uk/clustalw). Login to comment 303 ABCC2 p.Cys1515Tyr ABCC2 p.Val1188Glu V1188E/C1515Y variants showed the greatest alterations in function and/or expression in the ScIn in vitro system. Login to comment