ABCMdb

PMID: 15743976

Vethanayagam RR, Wang H, Gupta A, Zhang Y, Lewis F, Unadkat JD, Mao Q
Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N.
Drug Metab Dispos. 2005 Jun;33(6):697-705. Epub 2005 Mar 2., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Login to comment 4 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. Login to comment 5 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. Login to comment 7 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Login to comment 8 ABCG2 p.Asp620Asn Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. Login to comment 9 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. Login to comment 10 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP. Login to comment 20 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Two variants, V12M and Q141K, occurred at particularly high allele frequencies in Chinese and Japanese populations (30-60%) and at relatively lower allele frequencies in white people and African-Americans (5-26%) (Zamber et al., 2003). Login to comment 21 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Other variants such as I206L, N590Y, and D620N are generally much less frequent, with allele frequencies of approximately 1% or less (Honjo et al., 2002; Zamber et al., 2003). Login to comment 28 ABCG2 p.Gln141Lys 6 Copyright (c) 2005 by The American Society for Pharmacology and Experimental Therapeutics 3657/3131752 DMD 33:697-705, 2005 Printed in U.S.A. 697 Q141K seems to be associated with reduced protein expression compared with wild-type BCRP in mammalian expression systems (Imai et al., 2002; Kondo et al., 2004). Login to comment 29 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Functional analysis of the I206L, N590Y, and D620N variants of BCRP has not been reported so far. Login to comment 30 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn In the present study, we stably expressed wild-type BCRP and the variants I206L, N590Y, and D620N in HEK cells and analyzed the effects of the variants on BCRP expression, plasma membrane localization, transport, and ATPase activities, as well as drug resistance characteristics. Login to comment 45 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The variants I206L, N590Y, and D620N were then generated using the QuickChange Site-directed Mutagenesis Kit (Stratagene) and the pBluescript SK(ϩ) plasmid carrying wild-type BCRP cDNA as a template, according to the manufacturer`s instructions. Login to comment 47 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn The primer pairs for I206L were 5Ј-CTTATCACTGATCCTTCCCTCTTGTTCTTGGATGAG-3Ј and 5ЈCTCATCCAAGAACAAGAGGGAAGGATCAGTGATAAG-3Ј; the primer pairs for N590Y were 5Ј-GA- ATTTTTGGGACAATACTTCTGCCCAGGACTC-3Ј and 5Ј-GAGTCCT- GGGCAGAAGTATTGTCCCAAAAATTC-3Ј; and the primer pairs for D620N were 5Ј-GTAAAGCAGGGCATCAATCTCTCACCCTGGGGC-3Ј and 5Ј-GCCCCAGGGTGAGAGATTGATGCCCTGCTTTAC-3Ј. Login to comment 128 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Results Stable Expression of Wild-Type BCRP and the I206L, N590Y, and D620N Variants in HEK Cells. Login to comment 129 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn To examine the effects of I206L, N590Y, and D620N on protein expression, plasma membrane localization, and function of BCRP, we generated the three variants by site-directed mutagenesis. Login to comment 133 ABCG2 p.Ile206Leu ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn Figure 1A shows a typical immunoblot of whole cell lysates prepared from various cell lines (482R-13, 482R-21, I206L-13, I206L-20, N590Y-1, N590Y-13, D620N-9, and D620N-10) that express the highest levels of BCRP. Login to comment 134 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The cell lines 482R-21, I206L-13, N590Y-1, and D620N-9 were used in all subsequent experiments. Login to comment 136 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Expression levels of wild-type BCRP and its variants I206L, N590Y, and D620N in stably transfected HEK cells. Login to comment 143 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn A typical immunoblot of the plasma membrane preparations showed that I206L, N590Y, and D620N were expressed at levels of approximately 0.45-fold, 3.6-fold, and 2.4-fold those of the wild-type protein (482R), respectively (Fig. 1B). Login to comment 148 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The I206L, N590Y, and D620N Variants Were Predominantly Routed to the Plasma Membrane. Login to comment 149 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn To explore whether the variants might influence plasma membrane localization of BCRP, the 482R-21, I206L-13, N590Y-1, and D620N-9 cells were examined by immunofluorescent confocal microscopy. Login to comment 150 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Cells transfected with cDNAs of wild-type BCRP (482R-21) and the three variants (I206L-13, N590Y-1, and D620N-9) showed strong plasma membrane staining (Fig. 2), suggesting that, similar to wild-type BCRP, all three variants were predominantly routed to the plasma membrane. Login to comment 151 ABCG2 p.Asn590Tyr There appears to be some intracellular staining for the variants, particularly for N590Y. Login to comment 153 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn To further confirm cell surface expression of the variants, the 482R, I206L, N590Y, and D620N cells were incubated with the phycoerythrin-labeled anti-BCRP surface mAb 5D3 and the IgG negative control. Login to comment 158 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The relative levels of I206L, N590Y, and D620N on the cell surface were calculated from three independent experiments to be approximately 0.79-fold, 3.1-fold, and 1.3-fold those of wild-type BCRP, respectively. Login to comment 168 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Selected areas of HEK cells expressing wild-type BCRP (482R) and the variants I206L, N590Y, and D620N are shown. Login to comment 182 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The cells expressing I206L exhibited apparent efflux activities comparable to those of the cells expressing wild-type BCRP for all three fluorescence compounds tested, whereas the cells expressing N590Y and D620N showed higher efflux activities. Login to comment 184 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn After normalization, the efflux activities of I206L were approximately 2to 3-fold those of wild-type BCRP for the three fluorescent substrates, and the efflux activities were reduced by approximately 60 to 70% and 40 to 50% for N590Y and D620N, respectively (Table 1). Login to comment 195 ABCG2 p.Ile206Leu ABCG2 p.Ile206Leu The I206L cells showed apparent resistance levels to MX and topotecan comparable to those of the cells expressing wild-type BCRP; however, after normalizing to the BCRP levels, I206L demonstrated an approximately 2-fold increase in resistance to MX and topotecan compared with wild-type BCRP. Login to comment 196 ABCG2 p.Asn590Tyr In contrast, after normalization to the BCRP levels, N590Y conferred resistance to MX and topotecan at levels only approximately 30% of those of wild-type BCRP. Login to comment 197 ABCG2 p.Asp620Asn MX resistance conferred by D620N was decreased by approximately 50%, whereas its level of topotecan resistance remained essentially unchanged. Login to comment 201 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn A similar pattern of the effects of MX, prazosin, and FTC on the basal ATPase activities of I206L, N590Y, and D620N was observed. Login to comment 202 ABCG2 p.Ile206Leu ABCG2 p.Ile206Leu After normalizing to the BCRP levels, the basal ATPase activity of I206L and its ATPase activities in the presence of MX and prazosin were approximately 3 times higher than the respective activities of wild-type BCRP; however, the differences between the ATPase activities of I206L and those of wild-type protein were not statistically significant. Login to comment 203 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn In contrast, N590Y and D620N exhibited basal ATPase activities and ATPase activities in the presence of MX and prazosin that were approximately 30% and 50% of the wild-type activities, respectively. Login to comment 204 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Moreover, the basal ATPase activities of N590Y and D620N and their prazosin-stimulated ATPase activities were significantly different from the respective activities of wild-type BCRP (Fig. 6). Login to comment 207 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Imai et al. (2002) demonstrated that Q141K was expressed at a lower level in transfected murine fibroblast PA317 cells and conferred lower levels of drug resistance compared with wild-type BCRP, whereas V12M exhibited an expression level and drug resistance profiles very similar to those of wild-type protein (Imai et al., 2002). Login to comment 208 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met Another study (Mizuarai et al., 2004) showed that V12M and Q141K were expressed in the polarized LLC-PK1 porcine kidney cells at levels comparable to that of wild-type protein; however, both V12M and Q141K conferred significantly lower drug resistance than wild-type protein, accompanied with increased drug accumulation and decreased drug efflux. Login to comment 209 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met Further analysis of the mechanism of transport dysfunction revealed that the apical membrane localization of V12M was disrupted, whereas the expression and apical membrane localization of Q141K were not affected; however, the ATPase activity of Q141K was decreased. Login to comment 210 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met A recent study illustrated that V12M was expressed in HEK cells at levels similar to that of wild-type BCRP, whereas the Q141K level was significantly decreased compared with wild-type protein; however, the transport activity normalized by the BCRP expression was almost the same for V12M, Q141K and wild-type BCRP (Kondo et al., 2004). Login to comment 212 ABCG2 p.Gln141Lys However, the lower Q141K expression appears to be consistent with in vivo data. Login to comment 213 ABCG2 p.Gln141Lys When 99 Japanese placental samples were analyzed, the subjects who were homozygous for Q141K were found to express significantly lower amounts of BCRP (Kobayashi et al., 2005). Login to comment 214 ABCG2 p.Gln141Lys The lower protein expression and/or transport dysfunction of Q141K could affect drug disposition. Login to comment 215 ABCG2 p.Gln141Lys A recent clinical study has shown that Q141K is associated with significant changes in pharmacokinetics of diflomotecan (Sparreboom et al., 2004). Login to comment 216 ABCG2 p.Gln141Lys In five patients heterozygous for Q141K, the plasma levels of diflomotecan after intravenous administration were 299% of those in 15 patients expressing wild-type BCRP. Login to comment 220 ABCG2 p.Gln166Glu ABCG2 p.Ser441Asn ABCG2 p.Ala149Pro ABCG2 p.Pro269Ser ABCG2 p.Arg163Lys Several other BCRP variants occurring at much lower allele frequencies (0.5-1%) such as A149P, R163K, Q166E, P269S, and S441N have also been characterized (Kondo et al., 2004). Login to comment 221 ABCG2 p.Ser441Asn Except for S441N, all of these BCRP variants showed protein expression, membrane localization, and transport function similar to those of wild-type protein (Kondo et al., 2004). Login to comment 222 ABCG2 p.Ser441Asn S441N exhibited impaired membrane localization and lower protein expression, indicating that this variant may also affect disposition of BCRP substrates. Login to comment 223 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn In the present study, we examined the I206L, N590Y, and D620N TABLE 1 FTC-inhibitable efflux activities of HEK cells expressing wild-type BCRP and its variants The FTC-inhibitable efflux activities of fluorescent compounds are represented by the differences (⌬F) in the median fluorescence between the FTC/efflux histograms and the efflux histograms. Login to comment 228 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn MX PhA BODIPY-Prazosin Rhodamine 123 (⌬F) ⌬F Ratio ⌬F Ratio ⌬F Ratio pcDNA 0 11.4 Ϯ 7.1 0 0 482R-21 42.5 Ϯ 8.4 1.0 121.9 Ϯ 27.5 1.0 127.0 Ϯ 51.5 1.0 0 I206L-13 52.8 Ϯ 3.0 2.76 131.7 Ϯ 17.3 2.40 127.2 Ϯ 80.2 2.23 0 N590Y-1 64.7 Ϯ 4.7* 0.42 149.3 Ϯ 22.2 0.34 147.5 Ϯ 97.1 0.32 0 D620N-9 67.1 Ϯ 7.1* 0.63 168.2 Ϯ 29.8* 0.55 149.1 Ϯ 68.7 0.47 0 * Indicates that the un-normalized ⌬F values for the 482R cells are significantly different (p Ͻ 0.05) from the N590Y or D620N cells as calculated by Student`s t test. variants. Login to comment 229 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn I206L, N590Y, and D620N were stably expressed in HEK cells. Login to comment 230 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The immunoblots of the plasma membranes revealed a markedly lower protein level for I206L compared with wild-type BCRP, whereas the levels of N590Y and D620N were increased (Fig. 1B). Login to comment 233 ABCG2 p.Ile206Leu The mechanism of lower expression for I206L is not known but might be related to the location of position 206 in the functionally important Walker B motif of the NBD in BCRP. Login to comment 235 ABCG2 p.Ile206Leu Likewise, the I206L variant may also affect maturation and hence decrease protein expression of BCRP. Login to comment 236 ABCG2 p.Ile206Leu ABCG2 p.Ile206Leu The apparent transport activities of the cells expressing I206L were not significantly changed (Fig. 4; Table 1); however, after normalization to the BCRP level, I206L exhibited efflux activities and drug resistance capabilities approximately 2 to 3 times higher than those of wild-type protein (Tables 1 and 2). Login to comment 237 ABCG2 p.Ile206Leu ABCG2 p.Ile206Leu One possible explanation for the increased transport activities of I206L is that the affinity of substrates to I206L and/or transport (turnover) efficiency of the variant is augmented. Login to comment 238 ABCG2 p.Ile206Leu Although not statistically significant, the ATPase activities of I206L were also found to be increased approximately 3-fold compared with wild-type protein (Fig. 6). Login to comment 239 ABCG2 p.Ile206Leu These results suggest that, similar to the mutants in the Walker B motif in MRP1, the I206L variant can reduce protein expression and influence BCRP activity. Login to comment 240 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The N590Y and D620N variants are predicted to be in the extracellular loop connecting the fifth and sixth TM segments of BCRP. Login to comment 241 ABCG2 p.Asn590Tyr Functional analysis showed that, after normalization to the BCRP level, N590Y exhibited less than 50% of wild-type efflux and drug resistance activities (Tables 1 and 2). Login to comment 242 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The efflux activities of D620N for MX, PhA, and BODIPY-prazosin were also reduced, but to a lesser extent as compared with N590Y (Table 1). Login to comment 243 ABCG2 p.Asp620Asn Whereas D620N conferred resistance to MX at a level approximately 50% of that of wild-type BCRP, its resistance to topotecan was essentially unchanged (Table 2). Login to comment 244 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn These data indicate that the naturally occurring N590Y and D620N mutations in the extracellular loop can alter function and substrate selectivity of BCRP without significantly affecting cell surface expression, and that a mutation such as D620N in BCRP could affect recognition of one substrate but not the other, likely due to the existence of different ligand binding sites on BCRP, which has been implicated in previous studies (Nakanishi et al., 2003; Gupta et al., 2004). Login to comment 247 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The cells expressing wild-type BCRP (Œ), the variants I206L (‚), N590Y (Ⅺ), and D620N (छ), and the vector control cells (F) were exposed to MX (A), topotecan (B), daunorubicin (C), and rhodamine 123 (D) at the various concentrations indicated. Login to comment 256 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn MX Topotecan Daunorubicin Rhodamine 123 IC50 RR Ratio IC50 RR Ratio IC50 RR IC50 RR nM nM nM nM pcDNA 32.5 Ϯ 3.1 8.5 Ϯ 1.0 34.2 Ϯ 4.1 5797.9 Ϯ 1209.4 482R-21 226.3 Ϯ 27.4 7.0 1.0 122.8 Ϯ 23.3 14.4 1.0 40.0 Ϯ 1.7 1.2 10281.0 Ϯ 1445.4 1.7 I206L-13 209.7 Ϯ 19.5 6.5 2.06 124.9 Ϯ 17.1 14.7 2.25 25.6 Ϯ 2.8 0.7 8829.4 Ϯ 1454.8 1.5 N590Y-1 287.9 Ϯ 24.0* 8.9 0.35 130.5 Ϯ 17.8 15.4 0.30 19.9 Ϯ 2.3 0.6 7867.9 Ϯ 3691.1 1.3 D620N-9 271.9 Ϯ 33.3* 8.4 0.48 278.3 Ϯ 18.9* 32.7 0.91 31.6 Ϯ 1.8 0.9 15469.3 Ϯ 1762.5 2.6 * Indicates that the un-normalized IC50 values of the 482R cells are significantly different (p Ͻ 0.05) from the N590Y or D620N cells as calculated by Student`s t test. ATPase activities of both N590Y and D620N were also decreased to approximately 30 to 50% of the wild-type activities (Fig. 6). Login to comment 258 ABCC2 p.Trp208Gly For instance, the W208G mutation located in the second extracellular loop of P-gp was shown to drastically reduce affinity of valinomysin, FK506, and ␣-factor for P-gp (Kwan and Gros, 1998). Login to comment 261 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Our data, demonstrating that the efflux activities and drug resistance capabilities of N590Y and D620N, except for the topotecan resistance, are decreased, indicate that amino acid changes in the extracellular loop of BCRP can selectively affect function of the transporter. Login to comment 265 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn However, the immunoblots of both whole cell lysates and the plasma membranes did not illustrate any reduction or increase in molecular weight of N590Y and D620N compared with wild-type protein (Fig. 1). Login to comment 266 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn These results rule out the possibility that N590Y and D620N would be involved in N-glycosylation. Login to comment 267 ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn Interestingly, ATPase activities of N590Y and D620N were coincidentally impaired as the transport activities, even though the two mutations are not within the NBD. Login to comment 268 ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu The ATPase activities of the G406L/G410L mutant, which is predicted in the first TM segment of BCRP, were found to be completely abolished (Polgar et al., 2004). Login to comment 269 ABCG2 p.Gln141Lys ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn ABCG2 p.Gly406Leu ABCG2 p.Gly410Leu These data suggest that, whereas ATPase activities of BCRP could be affected by mutations in the NBD (e.g., Q141K and I206L), mutations in other regions including the TM segments (e.g., G406L/G410L) and extracellular loops (e.g., N590Y and D620N) can also influence ATP hydrolysis. Login to comment 270 ABCG2 p.Ile206Leu The I206L variant was identified so far only in Hispanic livers with a 20% allele frequency from a small number of samples (Zamber et al., 2003). Login to comment 271 ABCG2 p.Asn590Tyr The N590Y variant was present in white people with approximately 0.6 to 1.5% allele frequencies (Zamber et al., 2003; Mizuarai et al., 2004). Login to comment 272 ABCG2 p.Asp620Asn The D620N variant was detected in 1.1% of all DNA samples examined with unknown genetic origin (Honjo et al., 2002). Login to comment 273 ABCG2 p.Ile206Leu ABCG2 p.Asn590Tyr ABCG2 p.Asp620Asn The in vitro analysis in this study revealed that, after normalization to the BCRP expression levels, the specific activities of I206L, N590Y, and D620N were significantly altered as compared with wild-type BCRP; however, the overall efflux activities and drug resistance profiles of the cells expressing these variants remained essentially unchanged. Login to comment