ABCMdb

ABCG2 p.Ser441Asn

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Publications

PMID: 15475413 [PubMed] Kobayashi D et al: "Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta."

No. Sentence Comment

110 Of these, five SNPs resulted in the following amino acid substitutions: G34A (Val12Met), C376T (Gln126stop), C421A (Gln141Lys), G1322A (Ser441Asn), and T1465C (Phe489Leu). Login to comment
112 C376T, which is associated with an amino acid substitution from Gln to a stop codon at codon 126 (Gln126stop), was detected in only two placental samples (1.0%) as TABLE 1 Genetic polymorphism in the BCRP gene in Japanese placentas (n ϭ 100) Location Positiona Reference Alleleb Variant Allele Amino Acid Substitution Genotype Frequency of Variant Allele R/R R/V V/V 5Ј-Flanking region -20445 gtctCctcc gtctTctcc 98 2 0 0.010 -20296 agctAttaa agctGttaa 80 18 2 0.110 -19781 aaaaAttat aaaaGttat 99 1 0 -19572_-19569 ctcaCTCAcaaa ctca--caaa 60 33 7 0.235 Exon 2 34 cccaGtgtc cccaAtgtc Val12Met 70 24 6 0.180 Intron 2 203 ϩ 16 tttaAttta tttaGttta 70 24 6 0.180 Intron 3 263 ϩ 10 tataAgaga tataGgaga 85 14 1 0.080 263 ϩ 72 ttttGtgtg ttttTGtgtg 99 1 0 0.005 Exon 4 376 ggtaCaagt ggtaTaagt Gln126stop 98 2 0 0.010 Exon 5 421 cttaCagtt cttaAagtt Gln141Lys 42 45 13 0.355 Intron 5 532-16 ttatAatat ttatGatat 99 1 0 0.005 Exon 9 1098 aggaGatca aggaAatca Synonymous 98 2 0 0.010 Intron 10 1277 ϩ 95 atagTgtaa atagAgtaa 97 3 0 0.015 Exon 11 1322 agcaGtgtt agcaAtgtt Ser441Asn 99 1 0 0.005 Intron 11 1367 ϩ 20 ttctAggaa ttctGggaa 71 25 4 0.165 Exon 12 1465 tataTttac tataCttac Phe489Leu 99 1 0 0.005 Intron 12 1492 ϩ 49 ctatGggtg ctatCggtg 44 45 11 0.335 Exon 13 1515 atgcCttct atgc-ttct Phe506Ser 99 1 0 0.005 Phe507Leu Val508Leu Met509stop Intron 13 1648-42 tgaaAttac tgaaTttac 99 1 0 0.005 1648-21 gactCttag gactTttag 71 25 4 0.165 Intron 14 1738-46 tcttAaaat tcttGaaat 24 52 24 0.500 3Ј-UTR 2332 cttcAgtct cttcTAgtct 86 14 0 0.070 2364 tgccAttat tgccCttat 99 1 0 0.005 2512 agaaCttac agaaTttac 99 1 0 0.005 R, reference allele; V, variant allele. Login to comment

PMID: 15553238 [PubMed] Kondo C et al: "Functional analysis of SNPs variants of BCRP/ABCG2."

No. Sentence Comment

3 The cellular localization was identified using the wild type and seven different SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP) after transfection of their cDNAs in plasmid vector to LLC-PK1 cells. Login to comment
6 Wild type and six different SNP variants of BCRP other than S441N BCRP were expressed on the apical membrane, whereas S441N BCRP showed intracellular localization. Login to comment
7 The expression levels of Q141K and S441N BCRP proteins were significantly lower compared with the wild type and the other five variants. Login to comment
10 These results suggest that Q141K SNPs may associate with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. Login to comment
26 On analyzing the specimens from the 100 Japanese volunteers, 7 kinds of SNPs were identified for the BCRP gene: G34A (V12M), C376T (Q376Stop), C421A (Q141K), G1098A (E366E), G1322A (S441N), T1465C (F489L), and C1515- (AFFVM505-509ASSL Stop). Login to comment
29 We constructed expression systems for the wild type and SNPs variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, S441N BCRP) and examined whether these SNPs variants of BCRP alter its localization, expression level, and transport activity. Login to comment
42 Using site-directed mutagenesis, SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S and S441N BCRP) were constructed on pcDNA3.1 vector (SNPs type BCRP/pcDNA3.1). Login to comment
49 S441N BCRP was amplified with 5Ј-CCAACCAGTGTTTCAGCAAT- GTTTCAGCCGTGGAACTC-3Ј and 5Ј-GAGTTCCACG- GCTGAAACATTGCTGAAACACTGGTTGG-3Ј. Login to comment
52 For SNPs type BCRPs, viruses were prepared in the same way, resulting in the production of pAd-SNPs BCRP (pAd-V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP). Login to comment
91 In our experimental system, except for one SNP variant of BCRP (S441N BCRP/pcDNA3.1), all variants showed the same localization as the wild-type BCRP, at the apical membrane of LLC-PK1 cells (Fig. 1). Login to comment
92 S441N BCRP was expressed intracellularly (Fig. 1). Login to comment
97 Except for two BCRP variants (Q141K and S441N BCRP), the expression levels of each BCRP SNPs were approximately the same as that of the wild-type BCRP (Fig. 2). Login to comment
98 The expression level of Q141K BCRP was approximately 30-40% of the wild-type BCRP, whereas that of S441N BCRP was much lower, and could not be determined with any accuracy (Fig. 2). Login to comment
110 Except for two SNP variants of BCRP (Q141K and S441N BCRP), the ATP-dependent uptakes per mg membrane protein of SNP variants (V12M, A149P, R163K, Q166E, P269S BCRP) were similar to that of the wild-type BCRP (Fig. 3a). Login to comment
111 The uptake activity of Q141K BCRP per mg membrane protein was approximately 30-40% of the wild-type BCRP, and that of S441N was almost the same as that of the GFP-infected control cells. Login to comment
113 Then, in order to compare the intrinsic transport activity of the wild-type and SNP variants of BCRP, the uptake determined per mg membrane protein (Fig. 3a) was normalized relative to the expression levels estimated by Western blot analysis (Fig. 2), except for S441N BCRP the expression level of which was extremely low (Fig. 2). Login to comment
120 Figure 5a shows the ATP-dependent uptake of DHEAS, PAH, and MTX per mg membrane protein for the wild-type and SNPs BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP). Login to comment
121 Although Q141K BCRP exhibited a lower activity than wild type BCRP, no significant transport was observed for S441N BCRP (Fig. 5a). Login to comment
127 The expression levels of wild type and S441N SNPs BCRP were also determined also in whole cell lysates. Login to comment
142 As far as the cellular localization was concerned, S441N BCRP was the only variant which was expressed in the intracellular compartment. Login to comment
143 We also found that the intracellular localization of S441N BCRP in HEK293 cells after transient expression (data not shown), whereas the wild-type BCRP was expressed on the cell membrane. Login to comment
145 Western blot analysis revealed that the expression of S441N is significantly lower than the wild type BCRP. Login to comment
184 Among the 100 Japanese specimens, S441N SNPs were only found in one heterozygous subject and, consequently, their allele frequency was calculated to be only 0.5%. Login to comment
188 Since pheophorbide a is also transported by human BCRP (10), it is likely that Q141K and S441N SNPs may be involved in the phototoxicity and protoporphyria induced by the intake of chlorophyll. Login to comment
190 These data suggest that the ability to protect stem cells from some genotoxic xenobiotics might be lower in subjects who have Q141K and S441N SNPs in BCRP gene. Login to comment
197 Since BCRP also transports SN-38 (28), it is possible that subjects who have Q141K or S441N SNPs variants of BCRP are more sensitive to SN-38. Login to comment
198 In conclusion, we have shown that two kinds of SNP variants of BCRP (Q141K and S441N BCRP) are associated with the reduced expression. Login to comment
199 In particular, S441N variation is associated with the altered cellular localization. Login to comment

PMID: 15743976 [PubMed] Vethanayagam RR et al: "Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N."

No. Sentence Comment

220 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 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 S441N exhibited impaired membrane localization and lower protein expression, indicating that this variant may also affect disposition of BCRP substrates. Login to comment

PMID: 16160819 [PubMed] Ishikawa T et al: "Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design."

No. Sentence Comment

113 These contradictory expression and localization data for ABCG2 variants indicate that differences in transfection conditions (transient or stable expression), the copy number of cDNA incorporated in genomic DNA or other cellular determinants may variably Table 2 Frequencies of ABCG2 alleles in different ethnic groups Position Ethnic group Variant allele Allele Reference Amino acid cDNA N Hetero Homo Frequency (%) V12M c.34G>A Japanese 29 9 1 19.0 Imai et al. (2002) Japanese 10 - - 15.0 Zamber et al. (2003) Japanese 220 61 8 17.5 Kobayashi et al. (2005) Chinese 10 - - 20.0 Zamber et al. (2003) Southeast Asians 10 - - 45.0 Zamber et al. (2003) Pacific Islanders 7 - - 64.0 Zamber et al. (2003) Swedish 60 2 0 1.7 B¨ackstr¨om et al. (2003) Dutch 100 11 1 6.5 Bosch et al. (2005) Caucasian 86 - - 2.0 Zamber et al. (2003) Caucasian 150 27 2 10.3 Mizuarai et al. (2004) Caucasian 150 11 0 3.7 Kobayashi et al. (2005) Ashkenazi Jewish 10 - - 10.0 Zamber et al. (2003) Middle Eastern 20 - - 5.0 Zamber et al. (2003) Africans North of Sahara 7 - - 14.0 Zamber et al. (2003) African American 150 17 1 6.3 Kobayashi et al. (2005) Mexicans 10 - - 10.0 Zamber et al. (2003) Hispanic Livers 5 - - 40.0 Zamber et al. (2003) Mexican Indians 5 - - 90.0 Zamber et al. (2003) Q126Stop c.376C>T Japanese 124 3 0 1.2 Imai et al. (2002) Japanese 60 2 0 1.7 Itoda et al. (2003) Japanese 220 4 0 0.9 Kobayashi et al. (2005) Caucasian 150 0 0 0.0 Mizuarai et al. (2004) Caucasian 150 0 0 0.0 Kobayashi et al. (2005) African American 150 0 0 0.0 Kobayashi et al. (2005) Q141K c.421C>A Japanese 124 48 9 26.6 Imai et al. (2002) Japanese 10 - - 35.0 Zamber et al. (2003) Japanese 220 90 27 32.7 Kobayashi et al. (2005) Chinese 95 43 11 34.2 de Jong et al. (2004) Chinese 10 - - 35.0 Zamber et al. (2003) Southeast Asians 10 - - 15.0 Zamber et al. (2003) Pacific Islanders 7 - - 14.0 Zamber et al. (2003) Swedish 60 10 1 10.0 B¨ackstr¨om et al. (2003) Dutch 100 20 2 12.0 Bosch et al. (2005) Caucasian 85 - - 14.0 Zamber et al. (2003) Caucasian 172 33 3 11.3 de Jong et al. (2004) Caucasian 150 22 2 8.7 Mizuarai et al. (2004) Caucasian 150 25 4 11.0 Kobayashi et al. (2005) Ashkenazi Jewish 10 - - 5.0 Zamber et al. (2003) Middle Eastern 20 - - 13.0 Zamber et al. (2003) Africans North of Sahara 7 - - 0.0 Zamber et al. (2003) African, Sub-Saharan 938 14 1 0.9 de Jong et al. (2004) African American 24 - - 0.0 Zamber et al. (2003) African American 150 5 1 2.3 Kobayashi et al. (2005) African American 94 8 1 5.3 de Jong et al. (2004) Mexicans 10 - - 5.0 Zamber et al. (2003) Hispanic Livers 5 - - 10.0 Zamber et al. (2003) Mexican Indians 5 - - 10.0 Zamber et al. (2003) R160Q c.479G>A Dutch 100 1 0 0.5 Bosch et al. (2005) I206L c.616A>C Japanese 10 - - 0.0 Zamber et al. (2003) Chinese 10 - - 0.0 Zamber et al. (2003) Southeast Asians 10 - - 0.0 Zamber et al. (2003) Pacific Islanders 7 - - 0.0 Zamber et al. (2003) Caucasian 65 - - 0.0 Zamber et al. (2003) Table 2 Continued Position Ethnic group Variant allele Allele Reference Amino acid cDNA N Hetero Homo Frequency (%) Ashkenazi Jewish 10 - - 0.0 Zamber et al. (2003) Middle Eastern 20 - - 0.0 Zamber et al. (2003) Africans North of Sahara 7 - - 0.0 Zamber et al. (2003) African American 15 - - 0.0 Zamber et al. (2003) Mexicans 10 - - 0.0 Zamber et al. (2003) Hispanic Livers 5 - - 10.0 Zamber et al. (2003) Mexican Indians 5 - - 0.0 Zamber et al. (2003) F431L c.1291T>C Japanese 60 1 0 0.8 Itoda et al. (2003) S441N c.1322G>A Japanese 100 1 0 0.5 Kobayashi et al. (2005) F489L c.1465T>C Japanese 60 1 0 0.8 Itoda et al. (2003) Japanese 100 1 0 0.5 Kobayashi et al. (2005) R575Stop c.1723C>T Dutch 100 1 0 0.5 Bosch et al. (2005) N590Y c.1768A>T Caucasian 65 - - 1.0 Zamber et al. (2003) Caucasian 150 1 0 0.3 Mizuarai et al. (2004) African Americans 15 - - 0.0 Zamber et al. (2003) D620N c.1858G>A Dutch 100 1 0 0.5 Bosch et al. (2005) affect the cellular processing and sorting of these proteins. Login to comment

PMID: 16259577 [PubMed] Sakurai A et al: "Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications."

No. Sentence Comment

210 In different ethnic groups, seven naturally-occurring non-synonymous SNPs have been reported: V12M, Q126Stop, Q141K, I206L, F431L, S441N, F489L, N590Y and D620N. Login to comment
213 Some of the above sequence variations showed an allele frequency of ~ 1% in distinct populations, Q126stop and F489L in the Japanese and N590Y in the Caucasian population [129-131,134,135], whereas most of the mutations were only detected in single individuals (e.g., I206L, F431L, S441N, D620N). Login to comment
225 Location Position Allele Amino acid Allele frequency in Caucasian populations Allele frequency in Japanese populatins Allele frequency in African populations n % n % n % Exon 2 34 G A 12 Val 12 Met 546 94.4 5.6 259 82.4 17.6 181 93.7 6.3 Exon 4 376 C T 126 Gln 126 stop 300 100 0 404 98.9 1.1 150 100 0 Exon 5 421 C A 141 Gln 141 Lys 717 89.0 11.0 354 69.4 30.6 1213 98.6 1.4 Exon 5 479 G A 160 Arg 160 Gln 100 99.5 0.5 ND ND ND ND ND ND Exon 11 1291 T C 431 Phe 431 Leu ND ND ND 60 99.2 0.8 ND ND ND Exon 11 1322 G A 441 Ser 441 Asn ND ND ND 100 99.5 0.5 ND ND ND Exon 12 1465 T C 489 Phe 489 Leu ND ND ND 160 99.4 0.6 ND ND ND Exon 14 1723 C T 575 Arg 575 stop 100 99.5 0.5 ND ND ND ND ND ND Exon 15 1768 A T 590 Asn 590 Tyr 215 99.5 0.5 ND ND ND 15 100 0 Exon 16 1858 T A 620 Asp 620 Asp 100 99.5 0.5 ND ND ND ND ND ND Data are from [129-135,137]. Login to comment
250 COOH H2N N590Y V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L F489L D620N R482G R482T S441N F571I EXTRACELLULAR INTRACELLULAR R160Q R575stop ATP-binding site (transient or stable expression), the copy number of cDNA incorporated in genomic DNA or other cellular determinants may variably affect the cellular processing and sorting of these proteins. Login to comment

PMID: 16337740 [PubMed] Cervenak J et al: "The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology."

No. Sentence Comment

109 To date, altogether eight non-synonymous (V12M, Q141K, I206L, F431L, S441N, F489L, N590Y, D620N), five synonymous (silent) (c.114TOC, c.369COT, c.474COT, c.1098GOA, c.1425AOG) missense mutations, one nonsense (Q126X), and one frameshift (c.1515delC) mutations were identified in the coding region of ABCG2 in healthy individuals or in patients [43-46,49,63-65]. Login to comment
112 Some of the above sequence variations showed an allele frequency of about 1% in distinct populations (Q126X, F489L in the Japanese and N590Y in the Caucasian population [45-47,49,64]), while most of the mutations were only detected in single individuals (missense mutations: I206L, F431L, S441N, D620N, and a frameshift mutation: c.1515delC [44-46,49]). Login to comment
147 In a recent study, similarly LLC-PKI cells where used to express the V12M and Q141K variants and additionally five other polymorphisms (A149P, R163K, Q166E, P269S and S441N [55]). Login to comment
148 Interestingly, they found that all polymorphisms, including V12M and Q141K, had an apical localization, and only the S441N variant showed intracellular staining. Login to comment
149 The impaired localization pattern of the S441N variant was accompanied by a poor expression level. Login to comment

PMID: 16402910 [PubMed] Krishnamurthy P et al: "Role of ABCG2/BCRP in biology and medicine."

No. Sentence Comment

301 Three of these resulted in the amino acid substitutions F431L, F489L, and S441N. Login to comment
302 Konda et al. extended these studies and suggested that the SNP resulting in the S441N substitution may affect the expression level and cellular localization of ABCG2 (152). Login to comment

PMID: 16608919 [PubMed] Tamura A et al: "Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport."

No. Sentence Comment

2 In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment
4 We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in porphyrin transport, whereas F489L exhibited impaired transport, approximately 10% of the activity observed for the wild type. Login to comment
6 Thus, among those genetic polymorphisms of ABCG2, at least the hitherto validated alleles of Q126stop, S441N, and F489L are suggested to be of clinical importance related to the potential risk of porphyria. Login to comment
36 We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, S441N, and F489L are defective or impaired in the transport of porphyrins, suggesting that those genetic polymorphisms in the ABCG2 gene may be related to the risk of certain diseases resulting from disruption of porphyrin homeostasis. Login to comment
82 GC indicates the percentage of guanine and cytosine contents in the PCR primer set. Tm shows the melting temperature (Tm) for each PCR primer set. Variant and Primers Primer Sequence (5Ј 3 3Ј) Primer Length GC Tm bases % °C V12M 33 39 55 Forward CGAAGTTTTTATCCCAATGTCACAAGGAAACAC Reverse GTGTTTCCTTGTGACATTGGGATAAAAACTTCG G51C 42 35 59 Forward ATCGAGTAAAACTGAAGAGTTGCTTTCTACCTTGTAGAAAAC Reverse GTTTTCGACAAGGTAGAAAGCAACTCTTCAGTTTTACTCGAT Q126stop 40 40 62 Forward GTAATTCAGGTTACGTGGTATAAGATGATGTTGTGATGGG Reverse CCCATCACAACATCATCTTATACCACGTAACCTGAATTAC Q141K 35 42 55 Forward CGGTGAGAGAAAACTTAAAGTTCTCAGCAGCTCTT Reverse AAGAGCTGCTGAGAACTTTAAGTTTTCTCTCACCG T153M 42 40 60 Forward CGGCTTGCAACAACTATGATGAATCATGAAAAAAACGAACGG Reverse CCGTTCGTTTTTTTCATGATTCATCATAGTTGTTGCAAGCCG Q166E 35 42 55 Forward GGATTAACAGGGTCATTGAAGAGTTAGGTCTGGAT Reverse ATCCAGACCTAACTCTTCAATGACCCTGTTAATCC I206L 36 44 59 Forward CTTATCACTGATCCTTCCCTCTTGTTCTTGGATGAG Reverse CTCATCCAAGAACAAGAGGGAAGGATCAGTGATAAG F208S 35 45 55 Forward TGATCCTTCCATCTTGTCCTTGGATGAGCCTACAA Reverse TTGTAGGCTCATCCAAGGACAAGATGGAAGGATCA S248P 35 40 55 Forward TTCATCAGCCTCGATATCCCATCTTCAAGTTGTTT Reverse AAACAACTTGAAGATGGGATATCGAGGCTGATGAA E334stop 35 31 55 Forward TCATAGAAAAATTAGCGTAGATTTATGTCAACTCC Reverse GGAGTTGACATAAATCTACGCTAATTTTTCTATGA F431L 28 60 62 Forward AGCTGGGGTTCTCCTCTTCCTGACGACC Reverse GGTCGTCAGGAAGAGGAGAACCCCAGCT S441N 34 47 59 Forward AACCAGTGTTTCAGCAATGTTTCAGCCGTGGAAC Reverse GTTCCACGGCTGAAACATTGCTGAAACACTGGTT F489L 46 34 62 Forward GAGGATGTTACCAAGTATTATACTTACCTGTATAGTGTACTTCATG Reverse CATGAAGTACACTATACAGGTAAGTATAATACTTGGTAACATCCTC F571I 36 47 61 Forward GTCATGGCTTCAGTACATCAGCATTCCACGATATGG Reverse CCATATCGTGGAATGCTGATGTACTGAAGCCATGAC N590Y 42 38 62 Forward CATAATGAATTTTTGGGACAATACTTCTGCCCAGGACTCAAT Reverse ATTGAGTCCTGGGCAGAAGTATTGTCCCAAAAATTCATTATG D620N 32 56 62 Forward GGTAAAGCAGGGCATCAATCTCTCACCCTGGG Reverse CCCAGGGTGAGAGATTGATGCCCTGCTTTACC veloped by using Western Lighting Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA) and detected by Lumino Imaging Analyzer FAS-1000 (Toyobo Engineering, Osaka, Japan). Login to comment
144 For this purpose, based on the currently available data on SNPs and acquired mutations, we generated variant forms (i.e., V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis. Login to comment
164 It is important to note that the variants Q126stop, F208S, S248P, E334stop, and S441N substantially lack transport activity for both hematoporphyrin and methotrexate. Login to comment
177 as the variants F208S, S248P, S441N, F431L, and F489L were expressed in Flp-In 293 cells. Login to comment
179 Flp-In-293 cells expressing S441N were photosensitive (Fig. 6), suggesting that the S441N variant could not extrude pheophorbide a from cells, which lead them to become photosensitive. Login to comment
184 To gain more insight into the association of ABCG2 variants with cellular resistance to anticancer drugs, we incubated Flp-In-293 cells expressing ABCG2 WT, F431L, S441N, or F489L in the presence of SN-38, mitoxantrone, doxorubicin, or daunorubicin at different concentrations as described under Materials and Methods. Table 3 summarizes the drug resistance profile of those variants-expressing cells. Login to comment
185 Both Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (F431L) cells were resistant toward SN-38 and mitoxantrone, whereas the resistance ratio of Flp-In-293/ABCG2 (S441N) and Flp-In-293/ABCG2 (F489L) cells were much lower, being close to that of Flp-In-293/Mock cells. Login to comment
186 None of the SNP variants of F431L, S441N, and F489L conferred Flp-In-293 cells resistance to doxorubicin or daunorubicin (Table 3), being different from the acquired mutants of R482G and R482T (Yoshikawa et al., 2004). Login to comment
203 Photosensitivity of Flp-In-293 cells expressing ABCG2 WT, F431L, S441N, or F489L. Login to comment
214 In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment
215 We provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in the transport of hematoporphyrin (Fig. 5). Login to comment
217 Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when cells were treated with pheophorbide a. Thus, it is likely that humans with these alleles may be more susceptible to porphyrin-induced phototoxicity. Login to comment
219 The frequencies of the Q126stop, S441N, and F489L alleles are relatively low (less than 2%) compared with those of the V12M and Q141K alleles. Login to comment
224 Potential Risk Amino Acid Transport Allele Frequency cDNA Position Located on Exon Allele Data Sourcea Hemato MTX Wild-Type Allele % V12M ϩϩ ϩϩ 2.0-90.0 34 2 G A 1, 2, 4, 5, 7, 8 ૽૽ Q126stop - - 0.0-1.7 376 4 C T 1, 3, 5, 7 Q141K ϩϩ ϩϩ 0.0-35.5 421 5 C A 1, 2, 4, 5, 6, 7, 8 T153M ϩϩ ϩϩ 3.3 458 5 C T 5 R160Q N.D. N.D. 0.5 479 5 G A 8 Q166E ϩϩ ϩϩ N.D. 496 5 C G NCBI dbSNP rs1061017 I206L ϩϩ ϩϩ 10.0 616 6 A C 2 ૽૽ F208S - - N.D. 623 6 T C NCBI dbSNP rs1061018 ૽૽ S248P - - N.D. 742 7 T C NCBI dbSNP rs3116448 ૽૽ E334stop - - N.D. 1000 9 G T NCBI dbSNP rs3201997 F431L ϩϩ - 0.8 1291 11 T C 3 ૽૽ S441N - - 0.5 1322 11 G A 7 ૽ F489L ϩ - 0.5-0.8 1465 12 T C 3, 7 F571L ϩϩ ϩϩ 0.5 1711 14 T A NCBI dbSNP rs9282571 (૽૽) R575stop N.D. N.D. 0.5 1723 14 C T 8 N590Y ϩϩ ϩϩ 0.0-1.0 1768 15 A T 2, 5 D620N ϩϩ ϩϩ 0.5 1858 16 G A 8 Hemato, hematoporphyrin; NCBI, National Center for Biotechnology Information; N.D., not determined; ૽, risk of porphyria; (૽), potential risk is assumed as the lack of transport activity being as a result of a truncated protein. Login to comment
227 TABLE 3 Drug resistance profiles of ABCG2 WT and variants The drug resistance profiles of ABCG2 WT and variants were obtained by incubating Flp-In-293/ABCG2 WT, F431L, S441N, or F489L cells in the presence of SN-38, mitoxantrone, doxorubicin, or daunorubicin at different concentrations (0-100 ␮M) as described under Materials and Methods. Login to comment
233 Anticancer Drug IC50 and Drug Resistance Ratio Mock WT F431L S441N F489L nM (-fold) SN-38 0.9 Ϯ 0.1 (1.0) 42.1 Ϯ 3.1 (46.8)* 11.5 Ϯ 0.9 (12.7)* 0.7 Ϯ 0.1 (0.8) 3.1 Ϯ 0.3 (3.4) Mitoxantrone 5.2 Ϯ 0.3 (1.0) 99.8 Ϯ 4.5 (19.2)* 20.3 Ϯ 1.9 (4.4)* 4.6 Ϯ 0.5 (0.9) 11.5 Ϯ 0.4 (2.2) Doxorubicin 32.0 Ϯ 0.6 (1.0) 48.1 Ϯ 2.0 (1.5) 39.0 Ϯ 3.5 (1.2) 20.3 Ϯ 1.9 (0.6) 44.6 Ϯ 3.9 (1.4) Daunorubicin 9.5 Ϯ 1.2 (1.0) 17.8 Ϯ 3.9 (1.8) 14.1 Ϯ 0.5 (1.5) 12.1 Ϯ 0.2 (1.3) 16.3 Ϯ 0.9 (1.7) *P Ͻ 0.01. Login to comment
240 Therefore, at least, the validated alleles such as Q126stop, S441N, and F489L with a loss of porphyrin transport activity are at potential risk of diseases. Login to comment
244 As exemplified by the S441N variant of ABCG2, amino acid substitution at the 441 site caused dramatic changes in the substrate specificity of ABCG2 (Fig. 5). Login to comment
245 To be precise, the S441N variant completely lost transport activity for both hematoporphyrin and methotrexate (Fig. 5). Login to comment

PMID: 16702730 [PubMed] Maekawa K et al: "Genetic variation and haplotype structure of the ABC transporter gene ABCG2 in a Japanese population."

No. Sentence Comment

85 115Haplotype Structure in Human ABCG2 (from |1836 to |1175 bp upstream of the translational start site) of the basal promoter,30) and was suggested to inuence irinotecan pharmacokinetics.31) The frequencies of two well-known nonsynonymous SNPs, 34GÀA (Val12Met) and 421CÀA (Gln141Lys), were 0.192 and 0.319 in our study, which were comparable to those in Chinese (0.204 and 0.2220.350, respectively).20,27) However, the frequencies were much higher than those in Caucasians (0.020.065 and 0.080.15), African-Americans (00.09 and 00.05), and a Swedish population (0.02 and 0.1).18,19,21,23,27) Of other relatively rare nonsynonymous SNPs, 376CÀT (Gln126X), 1291TÀC (Phe431Leu), 1322GÀA (Ser441Asn), 1465TÀC (Phe489Leu), and 1515delC (Phe506SerfsX4) were already detected in a Japanese population by Itoda et al.17) andWor Kobayashi et al.,23) but not found in other ethnic groups. Login to comment
100 Because the two rare nonsynonymous variations, 1515delC (F506SfsX4) and 1322GÀA (Ser441Asn), were found in the same patient, they were statistically estimated to be linked with each other. Login to comment
141 The thick lines represent the combinations with frequencies over 10z, and the thin lines represent the combinations with frequencies of 1.0 to 9.9z. 118 Keiko MAEKAWA et al. haplotype harboring nonsynonymous SNPs, 1465TÀC (Phe489Leu) (*2), 1291TÀC (Phe431Leu) (*3), 1322GÀA (Ser441Asn)W1515delC (Phe506SerfsX) (*4), and 1723CÀT (Arg575X) (*5), respectively. Login to comment
155 These results are inconsistent with those obtained by Mizuarai et al. and Morisaki et al., in which the reduced drug resistance was not caused by the decreased protein expression.25,26) Kondo et al. have shown that the Ser441Asn variant was not localized to apical membranes, but remains intracellular in the transfected LLC-PK1 cells,24) suggesting its reduced activity. Login to comment

PMID: 16766035 [PubMed] Cascorbi I et al: "Role of pharmacogenetics of ATP-binding cassette transporters in the pharmacokinetics of drugs."

No. Sentence Comment

919 0.015 Exon 11 c. 1322 G>A S441N 0.005 IVS 11 1367 A>G ? Login to comment

PMID: 16877258 [PubMed] Wakabayashi K et al: "Human ABC transporter ABCG2 in xenobiotic protection and redox biology."

No. Sentence Comment

176 Based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in Sf9 insect cells. Login to comment
177 The variants Q126stop, F208S, S248P, E334stop, and S441N were found to be defective in the transport of hematoporphyrin (Tamura et al., 2006) (Table 2). Login to comment
179 Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when those cells were treated with pheophorbide a (Tamura et al., 2006). Login to comment

PMID: 17015488 [PubMed] Sarkadi B et al: "Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system."

No. Sentence Comment

997 In healthy individuals or patients, altogether eight nonsynonymous (V12M, Q141K, I206L, F431L, S441N, F489L, N590Y, D620N), five synonymous (silent) (c. Login to comment

PMID: 17093005 [PubMed] Enokizono J et al: "Involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion and intestinal efflux of troglitazone sulfate, the major metabolite of troglitazone with a cholestatic effect."

No. Sentence Comment

192 BCRP has some functional single nucleotide polymorphisms (SNP), such as C376T (Q126stop), C421A (Q141K), and G1322A (S441N). Login to comment

PMID: 17228519 [PubMed] Tamura A et al: "Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system."

No. Sentence Comment

4 While mRNAs of those integrated ABCG2 variants and wild type were evenly expressed in Flp-In-293 cells, the protein expression levels of F208S and S441N variants were found to be markedly low. Login to comment
48 Standard method for functional validation of ABCG2 SNPs Journal of Experimental Therapeutics and Oncology Vol. 6 2006 3 Plasma Membrane inside outside S S S homodimer A B CH2N COOH V12M Q141K F208S S248P F431L S441N F489L R482G R482T Acquired mutation Figure 1. Login to comment
67 PCR primers and conditions for site-directed mutagenesis to create variants of ABCG2 Variant Forward/Reverse Primer sequence (5` →→ 3`) Primer length % GC Tm (ºC) (F/R) primers (bases) V12M F CGAAGTTTTTATCCCAATGTCACAAGGAAACAC 33 39 55 R GTGTTTCCTTGTGACATTGGGATAAAAACTTCG Q141K F CGGTGAGAGAAAACTTAAAGTTCTCAGCAGCTCTT 35 42 55 R AAGAGCTGCTGAGAACTTTAAGTTTTCTCTCACCG F208S F TGATCCTTCCATCTTGTCCTTGGATGAGCCTACAA 35 45 55 R TTGTAGGCTCATCCAAGGACAAGATGGAAGGATCA S248P F TTCATCAGCCTCGATATCCCATCTTCAAGTTGTTT 35 40 55 R AAACAACTTGAAGATGGGATATCGAGGCTGATGAA F431L F AGCTGGGGTTCTCCTCTTCCTGACGACC 28 60 62 R GGTCGTCAGGAAGAGGAGAACCCCAGCT S441N F AACCAGTGTTTCAGCAATGTTTCAGCCGTGGAAC 34 47 59 R GTTCCACGGCTGAAACATTGCTGAAACACTGGTT F489L F GAGGATGTTACCAAGTATTATACTTACCTGTATAGTGTACTTCATG 46 34 62 R CATGAAGTACACTATACAGGTAAGTATAATACTTGGTAACATCCTC Sites of mutagenesis are indicated by underbars. Login to comment
104 Standard method for functional validation of ABCG2 SNPs Journal of Experimental Therapeutics and Oncology Vol. 6 2006 0 1 2 RelativemRNAlevel Mock WT V12M Q141K mRNA A ABCG2 GAPDH Mock WT F208S S248P F431L S441N F489L ABCG2 GAPDH 0 1 2 RelativemRNAlevel mRNA B GAPDH ABCG2 Mock WT F208S S248P F431L S441N F489L Protein 0 1 2 Relativeproteinlevel * * * C DProtein GAPDH ABCG2 0 1 2 Relativeproteinlevel * * Mock WT V12M Q141K Figure 3. mRNA and protein expression levels of ABCG2 WT and variants expressed in Flp-In-293 cells. Login to comment
114 Characterization of V12M, Q141K, F208S, S248P, F431L, S441N, and F489L variants expressed in Flp-In-293 cells The mRNA levels of ABCG2 and GAPDH were measured by quantitative PCR, and the ratios of ABCG2 variants vs. GAPDH were plotted. Login to comment
119 Figure 3 demonstrates mRNA and protein levels of ABCG2 WT and V12M, Q141K, F208S, S248P, F431L, S441N, and F489L variants expressed in Flp-In-293 cells. Login to comment
122 Interestingly, expression levels of the F208S and S441N variants were markedly low (Fig. 3D). Login to comment
123 The immunofluorescence images of Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells revealed that those variant proteins were not expressed in the plasma membrane (data not shown). Login to comment
132 Figure 4 summarizes the characteristics of those Tamura et al. 8 Journal of Experimental Therapeutics and Oncology Vol. 6 2006 Class Class Class Class WT V12M Q141K F431L S248P F489L F208S S441N R482G R482T Protein expression + + + + + + - - + + SN-38 resistance + + + + + / - - - - + + MX resistance + + + + / - - - - - + + Doxorubicin resistance - - - - - - - - + + Daunorubicin resistance - - - - - - - - + + Figure 4. Login to comment
140 Both F208S and S441N belong to the third class where protein expression levels were extremely low (Fig. 3D). Login to comment
142 Finally, the acquired mutants R482G and R482T form another group, which is characteristic Standard method for functional validation of ABCG2 SNPs Journal of Experimental Therapeutics and Oncology Vol. 6 2006 9 Table 3 Remarks mRNA Protein Author Ref Host cell Vector Expression SNP expression expression Imai et al. (15) PA317 pHaL-IRES-DHFR bicistronic Stable V12M Similar to WT Similar to WT - - retrovirus vector plasmid - Q141K Similar to WT Lower than WT Mizuarai et al. (18) LLC-PK1 pcDNA3.1(+) Stable V12M Similar to WT N.D. - - - - Q141K Similar to WT N.D. Morisaki et al. (25) HEK293 pcDNA3.1 Stable V12M Vary among clones Vary among clones - - - - Q141K Vary among clones Vary among clones - - - - D620N Vary among clones Vary among clones Kondo et al. (26) LLC-PK1/ pcDNA3.1/ Stable/ V12M N.D. Similar to WT - HEK293 Adenovirus Transient Q141K N.D. 30 - 40% of WT - - - - A149P N.D. Similar to WT - - - - R163K N.D. Similar to WT - - - - Q166E N.D. Similar to WT - - - - P269S N.D. Similar to WT - - - - S441N N.D. Lower than WT Vethanayagam (27) HEK293 pcDNA3.1/myc-His(-) Stable I206L N.D. Vary among clones et al. - - - - N590Y N.D. Vary among clones - - - - D620N N.D. Vary among clones N.D.: No data Table 2. Login to comment
143 Resistance profile (IC50 ) of ABCG2 Compounds IC50 (nM) Mock WT V12M Q141K F208S S248P F431L S441N F489L SN-38 1.0 ± 0.2 49.9 ± 6.0 51.1 ± 13.8 17.7 ± 0.9 0.7 ± 0.0 3.6 ± 0.4 12.1 ± 1.5 0.8 ± 0.0 3.9 ± 0.4 (49.9)* (51.1)* (17.7)* (0.7) (3.6) (12.1)* (0.8) (3.9) Mitoxantorone 7.0 ± 1.1 108.0 ± 4.9 94.0 ± 18.6 46.7 ± 12.7 5.1 ± 1.0 13.4 ± 1.3 15.2 ± 1.4 5.7 ± 0.8 12.1 ± 6.2 (15.4)* (13.4)* (6.7)* (0.7) (1.9) (2.2)* (0.8) (1.7) Doxorubicin 38.8 ± 3.8 105.2 ± 24.9 123.6 ± 35.3 156.8 ± 27.5 19.9 ± 8.7 23.7 ± 6.7 43.5 ± 6.1 39.4 ± 4.1 47.6 ± 3.1 (2.7) (3.2) (4.0) (0.5) (0.6) (1.1) (1.0) (1.2) Daounorubicin 13.0 ± 0.6 32.3 ± 6.5 58.2 ± 5.0 57.7 ± 4.1 14.1 ± 2.3 22.1 ± 4.2 15.9 ± 1.2 13.3 ± 1.1 23.6 ± 1.6 (2.5) (4.5) (4.4) (1.1) (1.7) (1.2) (1.0) (1.8) Etoposide 117.1 ± 16.0 210.2 ± 18.4 297.3 ± 58.5 233.9 ± 54.2 122.9 ± 17.6 137.7 ± 14.8 139.1 ± 12.3 154.3 ± 8.5 186.9 ± 10.1 (1.8) (2.5) (2.0) (1.0) (1.2) (1.2) (1.3) (1.6) Vincristine 1.8 ± 0.2 4.3 ± 0.3 7.1 ± 1.4 5.6 ± 1.6 0.6 ± 0.0 4.3 ± 0.9 1.8 ± 0.3 0.9 ± 0.1 3.0 ± 0.7 (2.4) (3.0) (3.1) (0.3) (2.4) (1.0) (0.5) (1.7) The drug resistance profiles of ABCG2 WT and variants were obtained by incubating Flp-In-293/ABCG2 WT, V12M, Q141K, F208S, S248P, F431L, S441N, or F489L cells in the presence of SN-38, mitoxantrone, doxorubicin, daunorubicin, etoposide, or vincristine at different concentrations as described in Materials and Methods. Login to comment

PMID: 17237154 [PubMed] Lee SS et al: "Identification and functional assessment of BCRP polymorphisms in a Korean population."

No. Sentence Comment

155 Recently, Kondo et al. (2004) reported the identification of several BCRP variants, which include A149P, R163K, Q166E, P269S, and S441N, in human cell lines. Login to comment

PMID: 17297656 [PubMed] Tamura A et al: "Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2."

No. Sentence Comment

3 To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Login to comment
6 In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Login to comment
8 The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Login to comment
137 Characterization of the F208S, S248P, F431L, S441N and F489L variants expressed in Flp-In-293 cells. Login to comment
138 Figure 2C demonstrates the mRNA and protein levels of WT ABCG2 and the F208S, S248P, F431L, S441N and F489L variants expressed in Flp-In-293 cells. Login to comment
139 Whereas mRNA levels were almost the same in WT and those variants, the protein levels of the F208S and S441N variants were markedly low. Login to comment
140 The immunofluorescence images of Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells revealed that those variant proteins were not expressed in the plasma membrane (Fig. 2D). Login to comment
141 The S441N variant appeared to remain in the intracellular space. Login to comment
145 Cells transfected with F 208S or S441N did not exhibit any resistance to SN-38 ormitoxantrone; their cell survival curves were very similar to that of Flp-In-293/Mock cells. Login to comment
176 Resistance profile (IC50) of ABCG2 Compound IC50 (nM) Mock Wild type V12M Q141K F208S S248P F431L S441N F489L SN-38 0.9 40.0 (44.4) 40.0 (44.4) 17.0 (18.9) 0.6 (0.7) 3.0 (3.3) 10.0 (11.1) 0.7 (0.8) 3.1 (3.4) Mitoxantorone 5.2 >100 (>19) 92.0 (17.7) 45.0 (8.7) 4.5 (0.9) 11.0 (2.1) 21.0 (4.0) 4.6 (0.9) 11.0 (2.1) Doxorubicin 32.0 78.0 (2.4) 100.0 (3.1) 110.0 (3.4) 20.0 (0.6) 20.0 (0.6) 40.0 (1.3) 21.0 (0.7) 45.0 (1.4) Daunorubicin 12.0 30.0 (2.5) 50.0 (4.2) 50.0 (4.2) 12.0 (1.0) 21.0 (1.8) 14.0 (1.2) 12.0 (1.0) 19.0 (1.6) Etoposide 110.0 200.0 (1.8) 220.0 (2.0) 200.0 (1.8) 110.0 (1.0) 120.0 (1.1) 120.0 (1.1) 130.0 (1.2) 170.0 (1.5) Vincristine 1.4 4.0 (2.9) 5.0 (3.6) 4.5 (3.2) 0.6 (0.4) 4.0 (2.9) 1.4 (1.0) 0.8 (0.6) 2.8 (2.0) Relative resistances to mock cells are described in parentheses. Login to comment
192 As clearly demonstrated in this study, the F208S, S248P, F431L, S441N and F489L variants exhibited greatly altered protein expression levels (Fig. 2C) or drug resistance profiles (Fig. 4 and Table 1). Login to comment
193 In particular, expression levels of the F208S and S441N variants were markedly low (Fig. 2C). Login to comment
202 As one of the specific aims of the present study, we functionally classified the non-synonymous polymorphisms (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) in terms of their protein expression level, drug resistance profile and prazosin-stimulated ATPase activity. Login to comment
207 Drug resistance profiles of Flp-In-293 cells expressing the wild-type (WT) BCRP/MXR1/ABCP (ABCG2), F208S, S248P, F431L, S441N or F489L variants toward (A) SN-38 and (B) mitoxantrone. Login to comment
214 (16) Both F208S and S441N belong to the third group where protein expression levels were extremely low (Fig. 2C). Login to comment

PMID: 17403009 [PubMed] Duncan EJ et al: "Cloning, mapping and association studies of the ovine ABCG2 gene with facial eczema disease in sheep."

No. Sentence Comment

95 The Q141K mutation affects the transport efficiency of the protein (Mizuarai et al. 2004), whereas the S441N mutation is known to alter the localisation of the mature protein from the cell membrane to an intracellular location. Login to comment

PMID: 17504223 [PubMed] Xia CQ et al: "Evaluation of drug-transporter interactions using in vitro and in vivo models."

No. Sentence Comment

119 The function of seven single nucleotide polymorphisms (SNPs) in BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP) was determined using membrane vesicles from HEK293 cells infected with the recombinant adenoviruses containing the corresponding BCRP cDNAs [45]. Login to comment
120 The expression levels of Q141K and S441N BCRP proteins were significantly lower compared to the wild type protein and the other five variants. Login to comment
122 These results suggest that Q141K SNPs may be associated with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. Login to comment

PMID: 18154452 [PubMed] Sharom FJ et al: "ABC multidrug transporters: structure, function and role in chemoresistance."

No. Sentence Comment

357 In a study of six different SNP variants, the C421A polymorphism (nonsynonymous, Q141K) was expressed at lower levels, and the S441N variant had both lower expression and altered localization [171]. Login to comment
368 A recent study characterized the activity of 18 ABCG2 variants, and concluded that Q126stop, F208S, S248P, E334stop, S441N and F489L are defective in hematoporphyrin transport [170], which may increase the risk of disease in individuals carrying these polymorphisms. Login to comment

PMID: 18159130 [PubMed] Tamura A et al: "In vitro evaluation of photosensitivity risk related to genetic polymorphisms of human ABC transporter ABCG2 and inhibition by drugs."

No. Sentence Comment

8 In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Login to comment
9 Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. Login to comment
22 By using plasma membrane vesicles and a high-speed screening system, we precisely evaluated functional changes associated with genetic polymorphisms in vitro.24) Since porphyrins are considered to be endogenous substrates for ABCG2, we have investigated the transport of porphyrins with a total of 18 variant forms of human ABCG2 in the plasma membrane vesicle system.4) As a result, we found that the variants Q126stop, F208S, S248P, E334stop, S441N, and F489L are defective or impaired in the transport of porphyrins. Login to comment
98 Genetic polymorphisms of human ABCG2 and pheophorbide a-photosensitivity In vitro experiments SNP data IC50 (mM) Photosensitivity ratio (fold) Ethnic group N Allele frequency (z) Reference WT 3.0 1.0 - - - - V12M 4.1 0.7 Caucasian 546 5.6 22, 13, 21, 20, 14 Japanese 259 17.6 18, 22, 20 African 181 6.3 22, 20 Q141K 2.9 1.0 Caucasian 717 11.0 22, 13, 21, 15, 20, 14 Japanese 354 30.6 18, 22, 20 African 1213 1.4 22, 15, 14 S441N 0.5 6.0 Japanese 100 0.5 20 F489L 1.7 1.8 Japanese 160 0.6 19, 20 Pheophorbide a-photosensitivity ratios and IC50 values were determined from the data shown in Fig. 2B. 432 Ai TAMURA, et al. a Fluoroskan Ascent FL (Thermo Labsystems, Helsinki, Finland) (excitation at 405 nm; emission at 612 nm). Login to comment
99 Results Expression of ABCG2 WT and SNP variants in Flp-In-293 cells: In the present study, we aimed to examine the impact of hitherto reported major SNPs (V12M, Q141K, S441N, or F489L) on the photo-sensitivity. Login to comment
107 Namely, the protein expression level of the S441N variant was markedly low (Fig. 1A). Login to comment
110 Figure 1B depicts the immuno‰uorescence images of Flp-In-293 cells expressing ABCG2 WT and those SNP variants (i.e., V12M, Q141K, S441N, and F489L) as well as mock vector-transfected cells (Flp-In-293/ Mock). Login to comment
115 In the case of the S441N variant, however, the variant protein was detected only within intracellular compartments in Flp-In-293 cells. Login to comment
119 As demonstrated in Fig. 2A, in the concentration range of up to 2.5 mM, the accumulation of pheophorbide a in Flp-In-293/ABCG2 (S441N) was high, being similar to that in Flp-In-293/Mock cells. Login to comment
120 In addition, the cellular pherophorbide a accumulation in Flp-In-293/ABCG2 (F489L) cells was about the same level as those in Flp-In-293/ABCG2 (S441N) and Flp-In-293/Mock cells at the concentration of 2.5 mM. Login to comment
123 Expression of human ABCG2 WT, V12M, Q141K, S441N, and F489L in Flp-In-293 cells. Login to comment
129 433Genetic Polymorphisms of ABCG2 and Photosensitivity Risk whereas the S441N variant and F489L appeared unable to export pheophorbide a. Login to comment
130 Photosensitivity of Flp-In-293 cells expressing ABCG2 WT and SNP variants: Figure 2B demonstrates the cellular photosensitivity proˆles of Flp-In-293 cells expressing ABCG2 WT, V12M, Q141K, S441N, and F489L, as well as that of Flp-In-293/Mock cells. Login to comment
136 Flp-In-293/Mock and Flp-In-293/ABCG2 (S441N) cells were very sensitive to light, whereas Flp-In-293/ABCG2 (V12M), Flp-In-293/ABCG2 (Q141K), and Flp-In-293/ABCG2 (WT) cells were signiˆcantly more resistant. Login to comment
141 A, Flp-In-293 cells expressing human ABCG2 WT and SNP variants (V12M, Q141K, S441N, and F489L) were incubated with pheophorbide a at diŠerent concentrations (0, 0.63, 1.25, and 2.5 mM) at 379C for 4 hours. Login to comment
199 Indeed, we reported that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in the transport of hematoporphyrin.4) The F489L variant showed impaired transport activity. Login to comment
200 As demonstrated in the present study, as well as in our previous one,4) Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when cells were treated with pheophorbide a. Thus, it is likely that humans with these alleles may be more susceptible to porphyrin-induced phototoxicity. Login to comment

PMID: 18237272 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2."

No. Sentence Comment

2 Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Login to comment
3 Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Login to comment
4 Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Login to comment
5 Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Login to comment
7 The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. Login to comment
26 The ABCG2 non-synonymous SNP variants Q126stop, F208S, S248P, E334stop, S441N and F489L were defective in the active transport of methotrexate and haematoporphyrin [18]. Login to comment
27 Furthermore, the F208S, S248P, F431L, S441N, and F489L ABCG2 variants exhibited greatly altered protein expression levels and drug Abbreviations used: ABC, ATP-binding cassette; ABCG2, ABC subfamily G, member 2; BMA, bafilomycin A1; CPT, camptothecin; DMEM, Dulbecco`s modified Eagle`s medium; endo H, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FCS, fetal calf serum; Flp, flippase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; ME, 2-mercaptoethanol; PNGase F, peptide N-glycosidase F; RT-PCR, reverse transcription-PCR; SN-38, 7-ethyl-10-hydroxycamptothecin; SNP, single nucleotide polymorphism; TBS, Tris-buffered saline; WT, wild-type. Login to comment
30 In particular, the expression levels of the F208S and S441N ABCG2 variant proteins were markedly low. Login to comment
32 Nevertheless, the mechanism underlying the low expression levels of those ABCG2 variants (i.e. F208S and S441N) has not yet been elucidated. Login to comment
39 We provide direct evidence that ABCG2 non-synonymous SNP variants, i.e., F208S and S441N, undergo ubiquitin-mediated protein degradation in proteasomes. Login to comment
45 Flp-In-293 cells expressing ABCG2 WT (wild-type), F208S or S441N Flp-In-293 cells expressing ABCG2 WT, F208S or S441N, named Flp-In-293/ABCG2 (WT), Flp-In-293/ABCG2 (F208S) or Flp-In-293/ABCG2 (S441N) respectively, were prepared as described previously [8,33]. Login to comment
88 To analyse quantitatively the distribution of the ABCG2 protein localized on the plasma membrane or in the cytosol, the immunofluorescence images captured by the confocal fluorescence microscopy system were processed by means of originally-developed computer software, Figure 1 Schematic illustration of human ABCG2 and expression of ABCG2 WT, F208S and S441N in Flp-In-293 cells at the transcription and protein levels (A) Arrows indicate the positions of amino acid substitutions in the non-synonymous SNP variants of ABCG2 F208S and S441N. Login to comment
93 (B) The mRNA level was analysed by RT-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT (WT), F208S or S441N. Login to comment
94 For comparison of the protein levels, the cell lysates of Flp-In-209 cells expressing ABCG2 WT (WT), F208S or S441N were analysed by immunoblotting with the ABCG2-specific monoclonal antibody (BXP-21, top panel) or the GAPDH-specific antibody after PNGase F treatment. Login to comment
102 RESULTS Protein expression levels of ABCG2 WT, F208S, and S441N in Flp-In-293 cells Figure 1(A) depicts a schematic illustration of the human ABCG2 protein in order to show the sites of amino acid alteration in the ABCG2 SNP variants F208S and S441N. Login to comment
103 WT, F208S and S441N ABCG2 were individually expressed in Flp-In-293 cells by using the Flp (flippase) recombinase system [8,33]. Login to comment
104 As shown in Figure 1(B), mRNA levels of ABCG2 WT, as well as F208S and S441N variants, were evenly represented in Flp-In-293 cells, where the mRNA levels of ABCG2 and GAPDH were determined by RT-PCR. Login to comment
105 WT, F208S and S441N ABCG2, as well as GAPDH, were also detected by immunoblotting, and their expression levels were quantified. Login to comment
109 Although mRNA levels were almost identical in ABCG2 WT and the SNP variants (F208S and S441N), the protein levels of those SNP variants were markedly low (Figure 1B). Login to comment
111 Characterization of F208S and S441N variants expressed in Flp-In-293 cells To gain insight into the molecular nature of ABCG2 WT and the two SNP variants (F208S and S441N), we performed immunoblot analysis experiments with cell lysate samples under reduced or non-reduced conditions. Login to comment
112 Figure 2(A) shows the effect of ME treatment on the migration of ABCG2 WT, F208S and S441N proteins on SDS/PAGE. Login to comment
115 These results suggest that the ABCG2 F208S and S441N proteins form homodimers through a cysteinyl disulfide bond, as observed for the WT ABCG2 protein. Login to comment
118 On the other hand, for the S441N ABCG2 variant, two forms of ABCG2 were detected at a molecular mass of approx. 81 kDa. Login to comment
122 Although the 81 kDa major band of ABCG2 WT was not at all affected by endo H treatment, the apparent molecular masses of the smaller protein bands of ABCG2 F208S (74 kDa) and S441N (78 kDa) decreased after endo H treatment. Login to comment
124 Among ABCG2 WT and the SNP variants Figure 2 Immunoblot detection of ABCG2 WT, F208S and S441N proteins expressed in Flp-In-293 cells (A) Effect of ME on the homodimer or monomer form of ABCG2 WT and the SNP variants. Login to comment
125 Cell lysate samples (20 µg of protein) were subjected to SDS/PAGE after treatment with [ME (+)] or without [ME(-)] ME. ABCG2 WT, F208S and S441N proteins were then detected by immunoblotting with the BXP-21 antibody as described in the Materials and methods section. Login to comment
126 After ME treatment ABCG2 WT, F208S and S441N proteins were converted from homodimers into monomers. Login to comment
131 ABCG2 WT, F208S and S441N proteins in the resulting samples were analysed by immunoblotting with the BXP-21 antibody. Login to comment
136 F208S and S441N, their N-linked oligosaccharide structures appear to be different. Login to comment
137 The mature N-linked oligosaccharide of ABCG2 WT may have a structure which is resistant to endo H, whereas the immature N-linked oligosaccharides of the F208S and S441N variant proteins are susceptible to this endoglycosidase. Login to comment
138 Effect of MG132 and BMA on the protein expression levels of F208S and S441N variants We hypothesize that the low protein expression levels of the F208S and S441N variants are due to rapid degradation of those proteins through ubiquitin-mediated proteasomal proteolysis. Login to comment
140 Flp-In-293 cells expressing ABCG2 F208S or S441N were incubated in the presence of 0, 0.4 or 2.0 µM MG132 for 24 h, and then cell lysate samples were immediately prepared. Login to comment
141 Protein expression levels of the F208S and S441N variants were determined by immunoblotting after PNGase F treatment as described above. Login to comment
144 After 24 h treatment with 2 µM MG132, ABCG2 F208S and S441N Figure 3 Effects of MG132 on the glycosylation status and protein levels of ABCG2 F208S and S441N variants expressed in Flp-In-293 cells (A) Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4 and 2 µM for 24 h at 37◦C. Cell lysate samples (20 µg of protein) were prepared in the presence of ME. ABCG2 F208S and S441N variant proteins were either analysed directly or treated with PNGase F for 10 min at 37◦C before immunoblotting with the ABCG2-specific monoclonal antibody (BXP-21). Login to comment
145 The signal intensity of the non-glycosylated form of the ABCG2 F208S or S441N variant was measured after PNGase F treatment. Login to comment
150 (B) Detection of aggregated forms (>200 kDa) of ABCG2 F208S and S441N variants. Login to comment
151 Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4 and 2 µM for 24 h at 37◦C. Cell lysate samples (20 µg of protein) were prepared in the presence of ME. ABCG2 F208S and S441N variant proteins were prepared from MG132-treated cells as described above and analysed by immunoblotting with the BXP-21 antibody in the absence of PNGase F treatment. Login to comment
152 The aggregated forms of ABCG2 F208S and S441N are indicated (arrowheads). Login to comment
160 It is of interest to note that MG132 treatments enhanced the levels of the non-glycosylated form (72 kDa) for both the F208S and S441N variants when those cell lysate samples were not treated with PNGase F (Figure 3A). Login to comment
161 More importantly, on immunoblot analysis without PNGase F treatment, aggregated forms (arrowheads in Figure 3B) of both the ABCG2 F208S and S441N variants were detected in the high-molecular-mass range (over 200 kDa). Login to comment
163 The protein level of ABCG2 WT increased more than 2.5-fold when cells were treated with BMA, which inhibits lysosomal degradation, whereas the ABCG2 F208S and S441N variant proteins were minimally affected by the same treatment. Login to comment
166 Effect of MG132 on the ubiquitination states of F208S and S441N variants To investigate the effect of MG132 on the ubiquitination states of the ABCG2 F208S and S441N variant proteins, Flp-In-293 cells expressing those SNP variants were incubated in the presence or absence of 2 µM MG132 for 24 h. Login to comment
167 As shown in Figure 4, significant increases in the ubiquitinated forms of the F208S and S441N variant proteins were detected by immunoblotting with mouse monoclonal anti-ubiquitin antibody after immunoprecipitation with anti-ABCG2 antibody (BXP-21). Login to comment
170 The corresponding results are presented in Figures 4(B) and 4(D), where ubiquitinated forms (arrowheads) appeared to be more prominent than with the monoclonal anti-ubiquitin antibody Figure 4 Effect of MG132 on ubiquitination of ABCG2 F208S and S441N Afterincubationinthepresenceorabsenceof2 µMMG132for24 hat37◦C,celllysatesamples were prepared from Flp-In-293/ABCG2 (F208S) (A, B) and Flp-In-293/ABCG2 (S441N) (C, D) cells. Login to comment
177 Effect of MG132 on the cellular localization of F208S and S441N variants It is of interest to know how inhibition of proteasomal protein degradation by MG132 affects the cellular localization of the ABCG2 F208S and S441N variant proteins. Login to comment
178 Figure 5 shows immunofluorescence images of Flp-In-293 cells expressing ABCG2 WT, F208S or S441N proteins that has been incubated in the presence or absence of 2 µM MG132 for 24 h. Login to comment
186 After Figure 5 Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 WT, F208S or S441N proteins Cells were incubated in the presence (+MG132) or absence (none) of 2 µM MG132 for 24 h at 37◦C. ABCG2 proteins were detected by using a mouse monoclonal ABCG2 antibody (either BXP-21 or 5D3) and Alexa Fluor® 488 (green fluorescence). Login to comment
190 In the case of the ABCG2 S441N variant, MG132 treatment enhanced the localization of this ABCG2 variant protein at the plasma membrane and in intracellular compartments (Figures 5j and 5l). Login to comment
195 Remarkable differences were observed after MG132 treatment in terms of the cellular localization and/or the quantity of the F208S or S441N variant ABCG2 proteins as shown in Figure 6(A). Login to comment
196 It is noteworthy that protein levels of the F208S and S441N variants were clearly enhanced in intracellular compartments after MG132 treatment, as observed with the BXP-21 antibody. Login to comment
197 In addition, plasma membrane localization of the S441N variant was also enhanced by MG132 treatment, as observed with the 5D3 antibody. Login to comment
201 Figure 6 Analysis of the effects of MG132 on the cellular localization of ABCG2 WT, F208S or S441N proteins in Flp-In-293 cells (A) Cells were incubated in the presence or the absence (None) of 2 µM MG132 for 24 h at 37◦C. ABCG2 proteins in cells were detected using an ABCG2-specific monoclonal antibody [either BXP-21 (BXP21) or 5D3] as described in the Materials and methods section. Login to comment
208 In a previous study using the Flp recombinase system [33], we functionally characterized the non-synonymous polymorphisms (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) in terms of their protein expression level, drug resistance profile and prazosin-stimulated ATPase activity. Login to comment
210 In particular, the expression levels of the F208S and S441N variant proteins were markedly low (Figure 1), consistent with the recent report of Yoshioka et al. [37]. Login to comment
212 Ubiquitin-mediated proteasomal degradation of F208S and S441N variants In the present study, we undertook the biochemical analysis of the molecular mechanisms underlying the low expression levels of the ABCG2 F208S and S441N variants. Login to comment
216 On the basis of those recent findings, we have examined the contribution of ubiquitin-mediated proteasomal degradation to the low-level expression of F208S and S441N ABCG2 variants by testing the effect of MG132. Login to comment
217 The presence of MG132 increased both the protein levels (Figure 3) and the ubiquitinated forms (Figure 4) of the F208S and S441N variant proteins. Login to comment
219 Misfolded proteins (e.g. ABCG2 F208S and S441N variants) are considered to be removed from the ER by retrotranslocation to the cytosol and then degraded by the ubiquitin-proteasome system by a process known as ERAD [38,39]. Login to comment
223 N-linked oligosaccharides of ABCG2 F208S and S441N variants During de novo synthesis in the ER, oligosaccharides are added to asparagine (N-glycosylation) or serine residues (O-glycosylation) of glycoproteins. Login to comment
231 Their findings were confirmed by recent experiments in which Asn596 of the ABCG2 protein was changed to glutamine and expressed in Flp-In-293 cells by using the Flp recombinase system (H. Nakagawa, Scheme 1 Schematic illustration of plausible pathways for protein processing and degradation of ABCG2 WT and SNP variants (F208S and S441N) We have provided evidence that ABCG2 WT and the variants undergo degradation by different pathways. Login to comment
236 It is important to note that N-linked oligosaccharide processing was greatly impaired in the ABCG2 F208S and S441N SNP variants. Login to comment
240 It is likely, however, that the ABCG2 F208S and S441N variant proteins did not undergo Golgi-apparatus-mediated glycoprocessing, but were passed through the so-called ERAD pathway. Login to comment
241 The immature and non-glycosylated forms of ABCG2 F208S and S441N were detected when Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were treated with MG132 for 24 h (Figure 3A), suggesting that those variant proteins were ubiquitinated in both pre-and post-N-glycosylation reactions and then readily degraded in proteasomes. Login to comment
244 While we have demonstrated here the ubiquitination and proteasomal degradation of the ABCG2 F208S or S441N variant homodimers, it will be important to study the fate of heterodimers (WT/SNP variant) in the future. Login to comment
251 As exemplified by the ABCG2 F208S and S441N variants, it is likely that several other SNP variants may undergo degradation via the ERAD pathway. Login to comment

PMID: 18249138 [PubMed] Hazai E et al: "Homology modeling of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

245 However, in our model, R482 cannot form interaction with rhodamine, but L484 is in interacting distance Table 3 Mutations on BCRP and their effect on its function Mutation Effect/results Reference V12M Did not effect Hemato and MTX transport Tamura et al. (2006) G51C Did not effect Hemato and MTX transport Tamura et al. (2006) K86M Inactivates transporter (dominant negative effect on ATPase activity); alters subcellular distribution Henriksen et al. (2005a) K86M Transporter inactive, but still able to bind ATP Ozvegy et al. (2002) Q126stop Defective porphyrin transport Tamura et al. (2006) Q141K Did not effect Hemato and MTX transport Tamura et al. (2006) T153M Did not effect Hemato and MTX transport Tamura et al. (2006) Q166E Did not effect Hemato and MTX transport Tamura et al. (2006) I206L Did not effect Hemato and MTX transport Tamura et al. (2006) F208S Defective porphyrin transport Tamura et al. (2006) S248P Defective porphyrin transport Tamura et al. (2006) E334stop Defective porphyrin transport Tamura et al. (2006) F431L Effects MTX transport Tamura et al. (2006) S441N Defective porphyrin transport Tamura et al. (2006) E446-mutants No drug resistance Miwa et al. (2003) R482G, R482T Effects MTX transport Tamura et al. (2006) R482T Substrate drug transport and inhibitor efficiency is not mediated by changes in drug-binding Pozza et al. (2006) R482G, R482T Substitution influence the substrate specificity of the transporter Ozvegy et al. (2002) R482G, R482T Altered substrate specificity Honjo et al. (2001) R482G Methotrexate not transported Chen et al. (2003b) Mitomo et al. (2003) R482G Resistance to hydrophilic antifolates in vitro, G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates Shafran et al., (2005) R482G Three distinct drug, binding sites Clark et al. (2006) R482G Altered substrate specificity, granulocyte maturation uneffected Ujhelly et al. (2003) R482 mutants Higher resistance to mitoxantrone and doxorubicin than wt Miwa et al. (2003) R482X Affects substrate transport and ATP hydrolysis but not substrate binding Ejendal et al. (2006) F489L Impaired porphyrin transport Tamura et al. (2006) G553L; G553E Impaired trafficing, expression, and N-linked glycosylation Polgar et al. (2006) L554P Dominant negative effect on drug sensitivity Kage et al. (2002) N557D Resistance to MTX, but decreased transport of SN-38; N557E no change in transport compared to wt Miwa et al. (2003) F571I Did not effect Hemato and MTX transport Tamura et al. (2006) N590Y Did not effect Hemato and MTX transport Tamura et al. (2006) C592A Impaired function and expression Henriksen et al. (2005b) C592A/C608A Restored plasma mb expression; MTX transport normal, BODIPY-prazosin impaired Henriksen et al. (2005b) C603A Disulfide bridge; no functional or membrane targeting change Henriksen et al. (2005b) C608A Impaired function and expression Henriksen et al. (2005b) D620N Did not effect Hemato and MTX transport Tamura et al. (2006) H630X No change in transport Miwa et al. (2003) Cand N-terminal truncated Impaired trafficing Takada et al. (2005) with the ligand. Login to comment

PMID: 18253130 [PubMed] Lemos C et al: "Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors."

No. Sentence Comment

45 Breedveld et al (2005) showed that Table 1 BCRP substrates and polymorphisms Substrates Classical anticancer drugs Novel targeted drugs Mitoxantrone Canertinib (CI-1033)a Anthracyclinesb Imatiniba Camptothecins Nilotiniba Antifolatesb Gefitiniba Erlotinib Flavopiridol Polymorphismsc Variant Amino-acid change Effect G34A Val12Met (V12M) No change C376T Gln126stop (Q126T) No active BCRP protein C421A Gln141Lys (Q141K) Decreased protein levels and drug resistance Increased gefitinib-associated toxicity (diarrhoea) Increased imatinib accumulation in vitro, but no changes in the pharmacokinetic parameters of imatinib in vivo G1322A Ser441Asn (S441N) Decreased protein levels and different subcellular localisation BCRP ¼ breast cancer resistance protein. Login to comment
113 Another SNP, G1322A (S441N), showed even lower levels of protein expression. Login to comment

PMID: 18363541 [PubMed] Tamura A et al: "Drug-induced phototoxicity evoked by inhibition of human ABC transporter ABCG2: development of in vitro high-speed screening systems."

No. Sentence Comment

230 Plasma membrane Outside Inside ATP-binding cassette H2 N COOH V12M G51C Q126stop Q141K T153M R160Q Q166E I206L F208S S248P E334stop F431L F489L S441N R482G R482T F571I R575stop N590Y D620N T542A A528T D296H P269S A. Login to comment
231 0.0 0.1 0.2 0.3 0.4 0.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T ATP-dependenthematoporphyrintransport (nmol/min/mgprotein) B. interactions should also take into consideration the presence of multiple flavonoids. Login to comment
245 Based on the presently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment
246 The variants Q126stop, F208S, S248P, E334stop, and S441N were defective in the transport of hematoporphyrin (Figure 9). Login to comment
248 Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when cells were treated with pheophorbide a [87,88]. Login to comment
249 To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed the cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that the accumulation of porphyrin and pheophorbide a in the cytoplasmic compartment was significantly higher in Flp-In-293 cells expressing S441N and F489L variants, as compared with those expressing WT, V12M, or Q141K [88]. Login to comment
252 Amino acid Porphyrin transport* Allele frequency (%)‡ cDNA position Location Wild-type allele Variant alllele V12M ++ 2.0 - 90.0 34 Exon 2 G A Q126stop - 0.0 - 1.7 376 Exon 4 C T Q141K ++ 0.0 - 35.5 421 Exon 5 C A T153M ++ 3.3 458 Exon 5 C T Q166E ++ N.D. 496 Exon 5 C G I206L ++ 10.0 616 Exon 6 A C F208S - N.D. 623 Exon 6 T C S248P - N.D. 742 Exon 7 T C E334stop - N.D. 1000 Exon 9 G T F431L ++ 0.8 1291 Exon 11 T C S441N - 0.5 1322 Exon 11 G A F489L + 0.5 - 0.8 1465 Exon 12 T C F571L ++ 0.5 1711 Exon 14 T A N590Y ++ 0.0 - 1.0 1768 Exon 15 A T D620N ++ 0.5 1858 Exon 16 G A *Transport of hematoporphyrin is indicated by either '+` (positive) or '-' (negative). Login to comment

PMID: 18464048 [PubMed] Gradhand U et al: "Pharmacogenomics of MRP transporters (ABCC1-5) and BCRP (ABCG2)."

No. Sentence Comment

250 It should be noted that many xeno- and endobiotic BCRP Figure 5 Predicted membrance topology of BCRP (ABCG2) based on hydrophobicity analysis. Locations of the non-synonymous polymorphisms are indicated with arrows. See Table 5 for allele frequencies and description of funtional consequences. NH2 COOH NBD Val12Met Gly51Cys Gln126* Ala149Pro Gln141Lys Thr153Met Arg160Gln Arg163Lys Gln166Glu Phe506Ser Phe507Leu Val508Leu Met509* Phe489Leu Ser441Asn Phe431Leu Glu334* Ile206Leu Ala315del Thr316del Phe208Ser Asp296His Ser248Pro Pro269Ser Phe571Ile Arg575* Asn590Tyr Asp620Asn in out Membrane BCRP (ABCG2) NBD Val12Met NBDNBD Val12Met substrates are also transported by other efflux transporters, especially P-glycoprotein, thus extrapolating BCRP related in vitro data to the in vivo situation may be difficult. Login to comment
287 Nevertheless, a number of groups have investigated the impact of the 1322G>T variation (Ser441Asn) and agree that this mutation leads to a markedly lower BCRP expression and loss in membrane localization in transfected cells (Kondo et al., 2004; Tamura et al., 2007). Login to comment
318 Available data in relation to BCRP pharmacogenetics suggest at least one of the known non-synonymous polymorphisms (421C>A/Gln141Lys) and possibly a few others (34G>A/Val12Met and 1322G>T/Ser441Asn) may be of functional and clinical importance. Login to comment

PMID: 18668433 [PubMed] Koshiba S et al: "Human ABC transporters ABCG2 (BCRP) and ABCG4."

No. Sentence Comment

225 Based on the currently available data on SNPs and acquired mutations, a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) were created by site-directed mutagenesis and expressed in Sf9 insect cells (Tamura et al. 2006, 2007). Login to comment
232 S. Koshiba et al. variants Q126stop, F208S, S248P, E334stop, and S441N substantially lack transport activity for both haematoporphyrin and methotrexate. Login to comment
235 Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when those cells were treated with pheophorbide a (Tamura et al. 2007). Login to comment
237 It has most recently revealed that F208S and S441N variant proteins were ubiquitinated and readily degraded in proteasomes in Flp-In-293 cells (Nakagawa et al. 2008). Login to comment

PMID: 18855611 [PubMed] Zhou SF et al: "Clinical pharmacogenetics and potential application in personalized medicine."

No. Sentence Comment

636 The localization of other variants including V12M, A149P, R163K, Q166E, P269S and S441N was also examined. Login to comment
637 All polymorphisms, including V12M and Q141K, had an apical localization, and only the S441N variant displayed an intracellular staining [275]. Login to comment

PMID: 18958403 [PubMed] Furukawa T et al: "Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations."

No. Sentence Comment

30 Furthermore, the Q141K SNP was reportedly associated with a higher incidence of diarrhea in non-small cell lung cancer patients treated with gefitinib (22). Login to comment
35 Furthermore, certain SNPs, such as F208S and S441N, were found to greatly affect the stability of ABCG2 in the endoplasmic reticulum (ER) and to enhance the protein degradation rate via ubiquitination and proteasomal proteolysis (32). Login to comment
60 The sites of three non-synonymous SNPs, Q141K, F208S and S441N, are indicated. Login to comment
129 Protein expression levels of the WT and the Q141K variant were determined by immunoblotting after PNGase F treatment in the same way as described above. Login to comment
130 As shown in Fig. 3A, the protein level of the Q141K variant was approximately two-fold enhanced by treatment with the proteasome inhibitor MG132. Login to comment
174 The nonsynonymous SNP variants of Q126stop, F208S, S248P, E334stop, S441N, and F489L were defective in the active transport of methotrexate and hematoporphyrin (42). Login to comment
175 Furthermore, the F208S, S248P, F431L, S441N, and F489L variants exhibited greatly altered protein expression levels and drug resistance profiles (18). Login to comment
176 In particular, expression levels of the F208S and S441N variant proteins were markedly low. Login to comment
131 In particular, expression levels of the F208S and S441N variant proteins were markedly low. Login to comment
344 The sites of three nonsynonymous SNPs, Q141K, F208S and S441N, are indicated. Login to comment

PMID: 19111842 [PubMed] Wakabayashi-Nakao K et al: "Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation."

No. Sentence Comment

813 Furthermore, certain non-synonymous single nucleotide polymorphisms (SNPs), such as Q141K, F208S, and S441N, were also found to greatly affect the stability of ABCG2 in the endoplasmic reticulum (ER) and to enhance the protein degradation rate via ubiquitination and proteasomal proteolysis [9a,b]. Login to comment
950 The non-synonymous SNP variants Q126stop, F208S, S248P, E334stop, S441N, and F489L were defective in the active transport of methotrexate and hematoporphyrin [54]. Login to comment
951 Furthermore, the F208S, S248P, F431L, S441N, and F489L variants exhibited greatly altered protein expression levels and drug resistance profiles [34]. Login to comment
952 In particular, expression levels of the F208S and S441N variant proteins were markedly low [9a,34]. Login to comment
953 5.2. Impact of SNPs on protein stability and function of ABCG2 Our recent study provided evidence that F208S and S441N variant proteins did not undergo Golgi apparatus-mediated glycoprocessing but were passed through the so-called "ERAD" pathway. Login to comment
954 The immature and non-glycosylated forms of F208S and S441N were detected when Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were treated with MG132 for 24 h, suggesting that those variant proteins were ubiquitinated in both pre and post N-glycosylation reactions and then readily degraded in proteasomes. Login to comment

PMID: 19200005 [PubMed] Porcelli L et al: "Intracellular trafficking of MDR transporters and relevance of SNPs."

No. Sentence Comment

149 Polymorphisms and Mutations Affecting the Cellular Localization of MDR Transporters Transporter Variant Amino-acid Change Localization References Intracellular + plasma membrane [99] C421A Q141K Plasma membrane [97, 98, 101] Intracellular + plasma membrane [98, 101] G34A V12M Plasma membrane [97, 99] ABCG2 G1322A S441N Intracellular [97, 98] ABCC1 G128C C43S Intracellular + plasma membrane [116] 4175-4180del RM1392-1393del Intracellular (ER) [118] C2302T R768W Intracellular (ER) [119] A3517T I1173F Intracellular (ER) [120, 121] C2366T S789F Intracellular + plasma membrane [122] ABCC2 G4348A A1459T Intracellular + plasma membrane [122] 293 transfected cells. Login to comment
194 Another polymorphism affecting the localization of the transporter is the G1322A nonsynonymous (S441N) SNP, which displayed intracellular localization in LLC-PK1 cells, contrasting with the apical localization of wild-type ABCG2 [97]. Login to comment
195 Additionally, the expression levels of S441N ABCG2 were much lower than the wild-type ABCG2 and also lower than the Q141K [97]. Login to comment
197 [98] showed that the S441N variant was associated with markedly lower levels of protein expression than the wild-type ABCG2, along with a marked increase in sensitivity to SN-38 and mitoxantrone. Login to comment
198 In contrast with the wild-type ABCG2, which was mainly expressed in the plasma membrane of the Flp-In-293 cells, the S441N variant appeared to remain in the intracellular space and was not expressed in the plasma membrane. Login to comment
217 From these studies it is clear that only the S441N and, possibly, the V12M and Q141K variants might affect ABCG2 localization within the cell. Login to comment

PMID: 19827267 [PubMed] Ishikawa T et al: "Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics."

No. Sentence Comment

222 COOH H2N N590Y V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L F489L D620N R482G R482T S441N F571I OUT IN R160Q R575stop ATP-binding site Figure 7. Continued A 005-024 pp JETO-0900616-TI (Review).indd 8/7/2009 3:59:50 19 Q141K has been associated with lower levels of protein expression and impaired transport in vitro (Imai et al., 2002; Kobayashi et al., 2005; Misuarai et al., 2004; Zamber et al., 2003; Morisaki et al., 2008; Kondo et al., 2004). Login to comment
227 The non-synonymous SNP variants Q126stop, F208S, S248P, E334stop, S441N, and F489L were defective in the active transport of methotrexate and hematoporphyrin (Tamura et al., 2006) (Fig. 7C). Login to comment
228 Furthermore, the F208S, S248P, F431L, S441N, and F489L variants exhibited greatly altered protein expression levels and drug resistance profiles Figure 7. Continued WT V12M Q141K F208S S248P F431L S441N F489L R482G R482T Protein expression + + + - + + - + + + MTX transport + + + - - - - +/ - - Porphyrin transport + + + - - + - +/ + + SN-38 resistance + + + - +/ + - - + + MX resistance + + + - - - - - -- - - - - - - - +/ - - - - - - - - + + Doxorubicin resistance + + Daunorubicin resistance + + ATPase activity (Prazosin) + + WTV12M Q141K F431L F489L S248P F208S S441L R482G R482T ∆1.5 ∆3 ∆3.5 ∆5 ∆4 - - - - - - -- - - B 005-024 pp JETO-0900616-TI (Review).indd 8/7/2009 3:59:51 20 Journal of Experimental Therapeutics and Oncology Vol. 8 2009 (Tamura et al., 2007b). Login to comment
229 In particular, expression levels of the F208S and S441N variant proteins were markedly low (Tamura et al., 2007b). Login to comment
230 We have recently shown that F208S and S441N variant proteins do not undergo Golgi apparatus-mediated glycoprocessing but are passed through the so-called "ERAD" pathway. Login to comment
231 The immature and non-glycosylated forms of F208S and S441N were detected when Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were treated with MG132 for 24 h, suggesting that those variant proteins were ubiquitinated in both pre and post N-glycosylation reactions and then readily degraded in proteasomes. Login to comment
232 It is known that, in the ER, the N-linked glycans play pivotal roles in protein fold- 0.0 0.5 1.0 1.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T Methotrexatetransport (nmol/min/mgprotein) Methotrexate 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T Methotrexatetransport (nmol/min/mgprotein) MethotrexateMethotrexate Porphyrintransport (nmol/min/mgprotein) 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 Porphyrin Figure 7. Login to comment

PMID: 19909340 [PubMed] Nakagawa H et al: "Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2."

No. Sentence Comment

22 In fact, the protein expression levels of ABCG2 SNP variants (Q141K, F208S and S441N) were significantly lower than that of the wild-type (WT) ABCG2 [23]. Login to comment

PMID: 19949922 [PubMed] Cascorbi I et al: "Pharmacogenetics of ATP-binding cassette transporters and clinical implications."

No. Sentence Comment

244 0.015 c. 1322 G>A S441N 0.005 c. 1367 A>G ? Login to comment

PMID: 19949928 [PubMed] Ross DD et al: "Impact of breast cancer resistance protein on cancer treatment outcomes."

No. Sentence Comment

84 transfected seven BCRP coding region SNPs into LLC-PK1 cells, and found lower BCRP -protein expression for the Q141K and S441N alleles (84). Login to comment
93 Tamura et al. used multicolor fluorescence in situ hybridization to assure uniform mRNA expression of cDNAs of seven BCRP SNPs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) transduced into Flp-In-293 cells (87, 88). Login to comment
94 Protein expression from the F208S and S441N variants was found to be low; the V12M and Q141K alleles had IC50 s for SN-38 that were approximately half that of the wild-type; all the other alleles examined had significantly lower IC50 values for SN-38, mitoxantrone, doxorubicin, daunorubicin and etoposide when compared with wild type alleles (88). Login to comment

PMID: 20700710 [PubMed] Wakabayashi-Nakao K et al: "Production of cells with targeted integration of gene variants of human ABC transporter for stable and regulated expression using the Flp recombinase system."

No. Sentence Comment

34 Furthermore, certain non-synonymous single nucleotide polymorphisms (SNPs), such as Q141K, F208S, and S441N, were also found to greatly affect the stability of ABCG2 in the ER and to enhance the protein degradation rate via ubiquitination and proteasomal proteolysis in Flp-In-293 cells (22-27) (Fig. 1). Login to comment
226 It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization of the F208S and S441N variant proteins (see Notes 5 and 6). Login to comment
227 Figure 6a depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 S441N that were incubated with or without 2 mM MG132 for 24 h. Login to comment
228 In the case of the S441N 3.3.4. Login to comment
245 Figure 3a depicts a schematic illustration of the human ABCG2 protein to indicate the sites of amino acid alteration in the SNP variants of F208S and S441N. Login to comment
247 Notes F208S, and S441N were individually expressed in Flp-In-293 cells by using the Flp recombinase system. Login to comment
248 As shown in Fig. 3b, mRNA levels of ABCG2 WT as well as F208S and S441N were evenly represented in Flp-In-293 cells, where the mRNA levels of ABCG2 and GAPDH were measured by RT-PCR. Login to comment
249 On the other hand, ABCG2 WT, F208S, and S441N as well as GAPDH proteins were detected by immunoblotting, and their expression levels were quantified. Login to comment
253 Schematic illustration of human ABCG2 as well as the expression of ABCG2 WT, F208S, and S441N in Flp-In-293 cells at the mRNA and protein levels. Login to comment
254 (a) Arrows indicate the positions of amino acid substitutions (Phe208Ser and Ser441Asn) in the non-synonymous SNP variants of F208S and S441N. Login to comment
259 (b) The mRNA level was analyzed by RT-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, F208S, or S441N. Login to comment
265 Although mRNA levels were almost the same in the WT and SNP variants (F208S and S441N), protein levels of those variants were markedly decreased (Fig. 3d). Login to comment
268 Figure 4a shows the effect of ME treatments on the SDS-PAGE migration of ABCG2 WT, F208S, and S441N proteins. Login to comment
270 Immunoblot detection of ABCG2 WT, F208S, and S441N proteins expressed in Flp-In-293 cells. Login to comment
271 (a) Effect of mercaptoethanol (ME) on the status (homodimer or monomer) of ABCG2 WT and the SNP variants. Cell lysate samples (20 mg of protein) were subjected to SDS-PAGE after treatments with or without ME; and, thereafter, ABCG2 WT, F208S, and S441N proteins were detected by immunoblotting with the BXP-21 antibody, as described in Subheading 3.3.3. Login to comment
272 By ME treatments, ABCG2 WT, F208S, and S441N proteins were changed from homodimers to monomers. Login to comment
273 (b) Effect of Endo H or PNGase F treatments on the glycosylated status of ABCG2 WT and the SNP variants. Cell lysate samples (20 mg of protein) were treated with Endo H or PNGase F, as described in Subheading 3.3.3.ABCG2 WT, F208S, and S441N proteins in the resulting samples were analyzed by immunoblotting with the BXP-21 antibody.The apparent molecular weights of mature and non-glycosylated ABCG2 WT were 81,000 and 72,000, respectively. Login to comment
280 These results provide evidence that F208S and S441N proteins form homodimers through a cysteinyl disulfide bond as does the ABCG2 WT protein. Login to comment
284 On the other hand, for the S441N variant, two protein bands of ABCG2 were detected around the molecular weight of 81,000. Login to comment
289 Although the major band (M.W. = 81,000) of ABCG2 WT was not at all affected by the Endo H treatment, the apparent molecular weights of the smaller protein bands of F208S (M.W. = 74,000) and S441N (M.W. = 78,000) decreased after the Endo H treatment. Login to comment
292 The matured N-linked oligosaccharide of ABCG2 WT may have a structure resistant to Endo H, whereas the immature N-linked oligosaccharides of the F208S and S441N variant proteins are susceptible to this endoglycosidase. Login to comment
295 Flp-In-293 cells expressing F208S or S441N were incubated in the presence of MG132 at different concentrations (0, 0.4, 2.0 mM) for 24 h, and then cell lysate samples were immediately prepared. Login to comment
296 Protein expression levels of the F208S and S441N variants were determined by immunoblotting after PNGase F treatments in the same way as described above. Login to comment
299 After the 24-h treatment with 2 mM MG132, F208S and S441N protein levels were increased 12-and 6-fold, respectively. Login to comment
301 Effects of MG132 on the glycosylation status and protein levels of ABCG2 F208S and S441N variants expressed in Flp-In-293 cells. Login to comment
302 (a) Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4, 2 mM for 24 h. Login to comment
304 ABCG2 F208S and S441N variant proteins were analyzed by immunoblotting with the ABCG2-specific monoclonal antibody (BXP-21) either directly or after PNGase F treatment.The signal intensity of the non-glycosylated form of the ABCG2 F208S or S441N variant was measured after PNGase F treatment.The intensities are represented as ratios relative to the ABCG2 level in MG132-untreated cells. Login to comment
308 (b) Detection of aggregated forms (M.W. > 200,000) of ABCG2 F208S and S441N variants. Login to comment
309 Flp-In-293/ABCG2 (F208S) and Flp-In-293/ ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4, 2 mM for 24 h. Login to comment
310 Cell lysate samples (20 mg of protein) were prepared in the presence of ME.ABCG2 F208S and S441N variant proteins were prepared from MG132-treated cells as described above and analyzed by immunoblotting with the BXP-21 antibody without PNGase F treatment. Login to comment
311 The aggregated forms of ABCG2 F208S and S441N are indicated by arrowheads. Login to comment
315 non-glycosylated form (M.W. = 72,000) for both F208S and S441N variants, where those cell lysate samples were not treated with PNGase F (Fig. 5a). Login to comment
316 More importantly, upon the immunoblot analysis without PNGase F treatments, aggregated forms (indicated by arrowheads in Fig. 5b) of both F208S and S441N variants were detected in the high molecular weight range of over 200,000. Login to comment
318 It was of great interest to know how the inhibition of proteasomal protein degradation by MG132 affects the cellular localization of the F208S and S441N variant proteins. Login to comment
319 Figure 6a depicts the immunofluorescence images of Flp-In-293 cells expressing ABCG2 S441N that were incubated with or without 2 mM MG132 for 24 h. Login to comment
320 In the case of the S441N variant, MG132 treatments enhanced the localization of the ABCG2 variant protein at the plasma membrane and in intracellular compartments. By immunoelectron microscopy, we could detect ABCG2 aggresome formation adjacent to the nuclei when Flp-In-293 cells expressing the F208 variant were treated with 2 mM MG132 for 24 h (Fig. 6b). Login to comment
323 Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 S441N protein (a) and immunoelectron microscopic image of Flp-In-293 cells expressing ABCG2 F208S protein (b). Login to comment

PMID: 20812902 [PubMed] Ni Z et al: "Structure and function of the human breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

249 A systematic study of 18 natural variants of BCRP expressed in insect cells showed that the variants Q126stop, F208S, S248P, E334stop, and S441N were defective in porphyrin transport, whereas F489L displayed approximately 10% of the transport activity of wild-type BCRP [120]. Login to comment

PMID: 21103975 [PubMed] Meyer zu Schwabedissen HE et al: "In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters (BCRP/MXR/ABCP/ABCG2)."

No. Sentence Comment

254 Reduced transport activity was also demonstrated for the c.1322G>A (p.S441N, AF 0.001 in Japanese population) variant. Login to comment

PMID: 21184741 [PubMed] Sugiyama T et al: "Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1."

No. Sentence Comment

29 Moreover, the certain single nucleotide polymorphism (SNP) variants of BCRP (Q141K, F208S and S441N), which protein expression was markedly low despite the functional expression of mRNA, were also degraded by ERAD [10,11]. Login to comment
162 These include naturally occurring SNPs variants of BCRP such as Q141K, F208S and S441N [10,11]. Login to comment

PMID: 21188243 [PubMed] Ishikawa T et al: "Key Role of Human ABC Transporter ABCG2 in Photodynamic Therapy and Photodynamic Diagnosis."

No. Sentence Comment

167 Based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells [41, 90]. Login to comment
168 The variants Q126stop, F208S, S248P, E334stop, and S441N are defective in the transport of hematoporphyrin (Figure 4(b)). Login to comment
170 Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when cells were treated with pheophorbide a. Login to comment
177 Gefitinib and imatinib are new anticancer drugs Outside Plasma membrane Inside H2N COOH V12M G51C Q126stop Q141K T153M R160Q Q166E I206L F208S S248P E334stop F431L F489L S441N R482G R482T F571I R575stop N590Y D620N T542A A528T D296H P269S ATP-binding cassette (a) 0 0.1 0.3 0.4 0.2 0.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T ATP-dependenthematoporphyrin transport(nmol/min/mgprotein) (b) Figure 4: (a) Schematic illustration of human ABCG2 and its nonsynonymous polymorphisms. Login to comment

PMID: 21567408 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts."

No. Sentence Comment

155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment
76 Furthermore, the nonsynonymous SNPs of Q141K, F208S, and S441N (Fig. 4) have been found to greatly affect APCG2 protein levels in the plasma membrane. Login to comment
78 Q141K (Gln141Lys), F208S (Phe208Ser), and S441N (Ser441Asn) are nonsynonymous SNPs that cause the reduced expression of ABCG2 protein. Login to comment
118 Impact of SNPs on Protein Stability and ERAD of ABCG2 By functional validation in vitro, the above-mentioned 17 nonsynonymous polymorphisms of ABCG2 were classified into four groups.99 The nonsynonymous SNP variants Q126stop, F208S, S248P, E334stop, S441N, and F489L were defective in the active transport of methotrexate and hematoporphyrin.100 The F208S, S248P, F431L, S441N, and F489L variants, on the contrary, exhibited greatly reduced protein expression levels and drug resistance profiles.99 In particular, expression levels of the F208S and S441N variant proteins were markedly low.99 These variant proteins do not undergo Golgi apparatus-mediated glycoprocessing but are passed through the ERAD pathway.78 The immature and nonglycosylated forms of F208S and S441N (Fig. 6a) were detected. Login to comment
119 When Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells were treated with MG132 for 24 h, the protein levels of F208S and S441N variants were greatly enhanced (Fig. 6a). Login to comment
128 Effects of MG132 on the glycosylation status and protein levels of ABCG2 F208S, and S441N variants expressed in Flp-In-293 cells. Login to comment
129 (a) Flp-In-293 cells expressing ABCG2 F208S or S441N variants were incubated with MG132 at concentrations of 0, 0.4, and 2 :M for 24 h. Cell lysate samples (20 :g of protein) were prepared in the presence of 2-mercaptethanol (ME). Login to comment
130 ABCG2 F208S, and S441N variant proteins were analyzed by immunoblotting with the ABCG2- specific monoclonal antibody (BXP-21) either directly or after PNGase F treatment. The signal intensity of the nonglycosylated form of the ABCG2 F208S or S441N variant was measured after PNGase F treatment. The intensities are represented as ratios relative to the ABCG2 level in MG132-untreated cells. Data are expressed as mean values ± SD in triplicate experiments (* p < 0.05). Login to comment
133 (b) Detection of aggregated forms (MW > 200,000) of ABCG2 F208S and S441N variants. Login to comment
134 Flp-In-293/ABCG2 (F208S) and Flp-In-293/ ABCG2 (S441N) cells were incubated with MG132 at concentrations of 0, 0.4, and 2 :M for 24 h. Cell lysate samples (20 :g of protein) were prepared in the presence of ME. Login to comment
135 ABCG2 F208S and S441N variant proteins were prepared from MG132-treated cells and analyzed by immunoblotting with the BXP-21 antibody without PNGase F treatment. The aggregated forms of ABCG2 F208S and S441N are indicated by arrowheads. Login to comment

PMID: 20103563 [PubMed] Klaassen CD et al: "Xenobiotic, bile acid, and cholesterol transporters: function and regulation."

No. Sentence Comment

6589 Absent C421A Q141K 2 Normal/reduced G445C A149P ↔ Normal G448A R163K ↔ Normal C496G Q166E ↔ Normal/reduced A616C I206L 2↔ Normal T623C F208S N.D. Reduced T742C S248P N.D. Normal C805T P269S 2↔ Normal T1291C F431L 2 Normal/reduced G1322A S441N 2 Reduced T1465C F489L 2↔ Normal/reduced A1768T N590Y 2↔ Increased G1858A D620N 2↔ Normal 2, reduced function; ↔, no change in function; N.D. not determined. Login to comment

PMID: 16180115 [PubMed] Ito K et al: "Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport."

No. Sentence Comment

212 Kondo et al. (119) also examined the cellular localization of a total of seven SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N) in LLC-PK1. Login to comment
213 As a result, reduced protein expression levels of Q141K and S441N were observed compared with the wild-type BCRP. Login to comment

PMID: 22248732 [PubMed] Natarajan K et al: "Role of breast cancer resistance protein (BCRP/ABCG2) in cancer drug resistance."

No. Sentence Comment

3488 Recently, evidence was presented that polymorphisms resulting in the variants F208S and S441N cause diminished BCRP protein levels by virtue of ubiquitin-mediated proteasomal degradation [100]. Login to comment

PMID: 22081364 [PubMed] Plante I et al: "Rosuvastatin blocks hERG current and prolongs cardiac repolarization."

No. Sentence Comment

24 Transfection Procedure In order to evaluate the effects of different transporters on the block of hERG by rosuvastatin, the hERG-stably transfected HEK 293 cells were transiently transfected with 10 :g of a recombinant pIRES2 vector expressing the green fluorescent protein (GFP) and one or the other of the following transporters: efflux transporter BCRP (strong affinity for rosuvastatin), loss of function variants of BCRP (BCRP-F208S, BCRP-S441N, and BCRP-Q141K), the influx transporters OATP2B1 (strong affinity for rosuvastatin), and multidrug resistance gene (MDR1) (an efflux transporter showing weak affinity for rosuvastatin). Login to comment
28 Site-Directed Mutagenesis The three loss-of-function variants of BCRP (BCRP-F208S, BCRP-S441N, and BCRP-Q141K) were Figure 1. Login to comment
88 Then, we performed site-directed mutagenesis on BCRP and produced three variants associated with a loss of function of the protein: C421A (Q141K), T623C (F208S), and G1322A (S441N).34-36 The coexpression of hERG with one or the other of these variants blunted the effects observed with the native efflux transporter BCRP, that is, the block was in the order of 40%. Login to comment
89 Indeed, inhibition of hERG was 40 ± 4 (n = 5), 41 ± 7 (n = 7), and 41 ± 4 (n = 7) for Q141K, F208S, and S441N, respectively (all not different from control, but different from HEK 293-hERG cells with functional transporters; p < 0.05). Login to comment
111 Inhibition of current activity was also determined in cells expressing transporters with a loss of function in BCRP activity due to mutations (BCRP-F208S, BCRP-S441N, and BCRP-Q141K). Login to comment
138 We also tested the effects of rosuvastatin on hERG in the presence of three loss-of-function polymorphisms of BCRP (Q141K, F208S, and S441N) and observed a block comparable to the one found in the control cells (≈42%). Login to comment

PMID: 22132966 [PubMed] Matsuo H et al: "ABCG2/BCRP dysfunction as a major cause of gout."

No. Sentence Comment

38 1119 ABCG2 : ATP binding casse e G2 SNP : single nucleo de polymorphism QTL : quan ta ve trait locus OR : odds ra o ABCG2 as a urate secre on transporter in humans Gene c analysis Func onal analysis ABCG2 muta on analysis of 90 hyperuricemic cases (all coding regions) ABCG2 muta ons (with amino acid altera ons) 6 muta ons c d Func onal analysis of urate transport via wild type ABCG2 (vesicle studies) a Iden fica on of urate transport ac vi es via ABCG2 b Func onal analysis of urate transport via mutated ABCG2 6 mutants e No effect (V12M) g Dysfunc onal genotype combina ons of ABCG2 as major causes of gout q Dysfunc onal SNP with high frequency (>30%) (Q141K) QTL analysis in 739 Japanese individuals h i j n Gout / hyperuricemia with ABCG2 homozygous, n = 2 heterozygous, n = 24 Loss of func on (Q126X, G268R, S441N, F506Sfs) Reduced func on (~50%) (Q141K) f p Genotype combina on analysis 10.1% of gout with ≤1/4 ABCG2 func on OR = 25.8, p = 3.39×10-21 o Haplotype analysis 13.5% of gout with disease haplotype OR = 5.97, p = 4.10×10-12 Associa on analysis of hyperuricemia (Q126X) OR = 3.61, p = 2.91× 10-7 l m Associa on analysis of gout (Q126X) OR = 4.25, p =3.04 × 10-8 Genotyping of nonfunc onal SNP (Q126X) hyperuricemia, n=228 k FIGURE 1 Flowchart for molecular-function-based clinicogenetic (FBCG) analysis of gout patients with ABCG2 polymorphic variants. Login to comment
53 Using the site-directed mutagenesis technique, we constructed ABCG2 mutants (V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4), which were used for urate transport analysis, on the expression vector for ABCG2. Login to comment
65 The following six nonsynonymous mutations, including three SNPs, were found: V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4 (Figure 2A). Login to comment
79 Among six mutants, ATP-dependent urate transport was reduced by approximately half (46.7%) in one mutant, Q141K, and was nearly eliminated in four mutants, Q126X, G268R, S441N, and F506SfsX4 (Figure 2B). Login to comment

PMID: 22132963 [PubMed] Matsuo H et al: "Identification of ABCG2 dysfunction as a major factor contributing to gout."

No. Sentence Comment

36 Using the site-directed mutagenesis technique, we constructed mutants of ABCG2 (V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4), which were used for urate transport analysis, on the expression vector for ABCG2. Login to comment
45 The following six non-synonymous mutations, V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4, were found (Figure 1A), and the first three mutations were SNPs. Login to comment
52 The ATP-dependent transport of urate was reduced by approximately half (46.7%) in Q141K and was nearly eliminated in Q126X, G268R, S441N, and F506SfsX4 mutants (Figure 1B). Login to comment

PMID: 22132962 [PubMed] Nakayama A et al: "ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans."

No. Sentence Comment

47 We found the following six nonsynonymous mutations: V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4, and the first three mutations are SNPs. Login to comment

PMID: 21554546 [PubMed] Woodward OM et al: "ABCG transporters and disease."

No. Sentence Comment

59 R L L A A M AT T T R V S G G G F I T Q R R V K K S G E A D RR V V K K L L G E E E I IN NN D H Q Q R V V V V V L L S G F E N M TT QD D S K R V K L L G F P C Y R K S G F P P C N N A V L L S G G G I N A D R K P P S GG G R V VK K KK L L L LL L S S S GG G PPE E IIII N NN M A A A T D D Y N E A I P E S I D L L F T LS G EI MT D I I P FC L R IH A N T T T T T G L D S S K K K L L L S G G G F F F F Q P P I M M A A A A D H G G LS S S V L L L L L R R RQ Q I I Y Y YS S HE E A T V V V V L Q I S F I I II A A L G G Y K F R S S E E I I L G Y YY Y V V K H S P C M M D R T I II L L L F F YV S S P F N T I A Q Q L L L G F Y Y H S S PR W C N M I I A A A L L G F V V K H W T L I F F C C C D D D A A A QQ Q Q Q G G G G G G G G G G FF FF F F FF Y Y Y Y Y V V V V V VVV K KKK KK K K E E E E P P P P R W W TT TT T TT T T NNNN N N N N N M MM M L L L L L L L L LL LL I I I I I I AA A A A A A S S S S S S S S SS L L L L LL L L LL V V F G GCC T Q Q Q Q Y Y Y KK K K K H H EE E E E E EEE E P P P P R R RW N N N II I I I I I I A AAA A A A S SS S S S S L L LL L L L V V V V F F F F F F F G GG G C TT T T T T K K K K KKKK N NN LL D DDD DS S 395 469 565 644 414 450 495 505 584 625 Signature Walker A WalkerBQ EP MI A V V VF FG GTN N NS S S S P F HE V FG CTT K NN LLD SS AAA I V12M N-terminus C-terminus M MM MM T A A A A L F F Y V V S S S F 524476 Y Q126X G268R S441N F506fs Q141K 44 288 PP AA DD Fig. 2. Login to comment

PMID: 20368174 [PubMed] Matsuo H et al: "Common defects of ABCG2, a high-capacity urate exporter, cause gout: a function-based genetic analysis in a Japanese population."

No. Sentence Comment

46 The following six nonsynonymous mutations were found: V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4 (Table 1). Login to comment
52 ATP-dependent transport of urate was reduced by approximately half (46.7%) in Q141K and was nearly eliminated in Q126X, G268R, S441N, and F506SfsX4 mutants (Fig. 2B). Login to comment
77 The call rate, or the ability of the SNP to be reliably decoded, for V12M, Q126X, and LS N N SV FLC S P T AN FK G LM ETS S E V F I P Q G N T N G FV P A A AS LD V S N I C Y R V K K RKPVEKEILSNINGIKPGLNAILGPG GGKSSL LDVLA ARKDP S G T L S G D V L I G A P PR A N F K N S G Y Q D D V V M G T L T V R NE LV VC H Q F S A A A RL L T T TNEKNER HINRVIQELGLDKVADSKVGTQFIRGVG GERR KTSIGME L I T D P S I L F L D E P T T G L D S S T A N A V LL L L K R M S K Q G R I I F S T S I H Q P R Y M S I F K LFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAN YN T V A L N R E E D F K A T E II E P S K Q D K L I E L A EK I Y V N S S F Y K ETKAELHQLSGGEKKKKITVFKEISYTTSFCHQRWVK SRS AFFLDII N G D S A PD P L F K N LL G N P Q A S A I V G I I T L V A FI I Q V V L G Y AVEFLKNDST G I Q N R A G V L F F L T T Q C F S L V S S N G L S L M L I T P M S F I FV D L R P I C Y W L W Y I Y T Q S R F L NQ S L F P G A H E F Y S Y S E F R G Y I K V K S V Y I H L E V A S S V L M A A M F V A F M S Y M T M F K A T I M L H F I A V K G W I L V C A N L W V A T L M T C F VI F M M I F S G L L VNLTTIASAIAAGQS L S V V LKGL L F N Q L F P S L D Y G Q K V L C Y EEGTCTAYNCPNNGTAN G P G L K L L L K K SYF L Y D L G L M A P K Extracellular Intracellular 50 150 200 300 100 350 395 415 469 450 470 500 525 550 565 585 600 625 608 650 250 655 603 475 644 F506SfsX4 (F506fs) V12M Q126X Q141K S441N G268R V Q F S G Q # C signature Walker B motif Walker A motif C D E 4.0 4.5 5.0 5.5 6.0 C/C C/A A/A Male + female P= 2.02 x 10 -6 5.0 5.5 6.0 6.5 7.0 C/C C/A A/A Male P= 0.0144 Serumuric acid(mg/dl) 4.0 4.5 5.0 5.5 6.0 C/C C/A A/A Female P= 0.0137 (pmol/mgprotein) 0 20 40 60 80 100 120 140 160 180 200 + AMP + ATP B Serumuric acid(mg/dl) Serumuric acid(mg/dl) A [C]Uratetransport 14 G F M C-terminal N-terminal Fig. 2. Login to comment
89 Amino acid change SNP ID dbSNP (NCBI) Exon Type of mutation Number of hyperuricemia patients Allele frequency (%) (in hyperuricemia) Allele frequency* (%) (in Japanese population) Wild-type Heterozygote Homozygote Q141K rs2231142 5 Missense 29 47 14 41.67 31.9 V12M rs2231137 2 Missense 64 23 3 16.11 19.2 Q126X 4 Nonsense 80 10 0 5.56 2.8 G268R 7 Missense 89 1 0 0.56 N.D. S441N 11 Missense 89 1 0 0.56 0.3 F506SfsX4 13 Frameshift 89 1 0 0.56 0.3 * Data from Maekawa et al. (34). Login to comment
181 Using the site-directed mutagenesis technique, we constructed mutants of ABCG2 (V12M, Q126X, Q141K, G268R, S441N, and F506SfsX4), which were used for urate transport analysis, on the expression vector for ABCG2. Login to comment

PMID: 16815813 [PubMed] Choudhuri S et al: "Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters."

No. Sentence Comment

573 Recently, Kondo et al. (2004) reported the effect of single nucleotide polymorphisms (SNPs) in ABCG2 gene on its localization, expression level, and transport activity of the BCRP protein. The cellular localization was identified using the wild-type and seven different SNP variants of BCRP protein (Val12Met, Gln141Lys, Ala149Pro, Arg163Lys, Gln166Glu, Pro269Ser, and Ser441Asn), following their expression in LLC-PK1 cells. Login to comment
575 The authors concluded that Gln141Lys variant of the BCRP protein may be associated with a lower expression level, and Ser441Asn variant may lower both the expression level and cellular localization. Login to comment

PMID: 25036722 [PubMed] Szafraniec MJ et al: "Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

201 To elucidate the significance of this polymorphism for porphyrin transport, a set of 18 variants of BCRP (Val12 Met, Gly51 Cys, Gln126 stop, Gln141 Lys, Thr153 Met, Gln166 Glu, Ile206 Leu, Phe208 Ser, Ser248 Pro, Glu334 stop, Phe431 Leu, Ser441 Asn, Arg482 Gly, Arg482 Thr, Phe489 Leu, Phe571 Ile, Asn590 Tyr and Asp620 Asn) have been expressed in insect cells. Login to comment
209 Position Type of mutation Effect on the transporter References NBD Lys 86 Met (i) No stimulation of the ATPase activity by prazosin; (ii) no influence on the transport of mitoxantrone Henriksen et al. (2005b) Glu 126 stop, Phe 208 Ser, Ser 248 Phe, Glu 334 stop Inability to transport hematoporphyrin Tamura et al. (2006) Glu 211 Gln Complete abolishment of the ATPase activity and methotrexate transport Hou et al. (2009) Pro 392 Ala Significant reduction in the efflux activity of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Ni et al. (2011) TM1 Gly 406 Ala Gly 410 Ala No influence on the activity of the transporter Polgar et al. (2004) Gly 406 Leu Gly 410 Leu (i) Loss of the ability to transport rhodamine123; (ii) impaired transport of mitoxantrone, Pheide and BODIPY-prazosin Polgar et al. (2004) Extracellular loop 1 Phe 431 Leu (i) Loss of the ability to transport methotrexate; (ii) 10% level of hematoporphyrin transport compared to the WT protein Tamura et al. (2006) Ser 441 Asn Inability to transport hematoporphyrin Tamura et al. (2006) Ser 441 Asn Loss of the ability to transport methotrexate Tamura et al. (2006) TM2 Lys 452 Ala His 457 Ala Increase in transport of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Cai et al. (2010) Lys 453 Ala Arg 465 Ala Decrease in transport of mitoxantrone, BODIPY-prazosin, Hoechst 33342, doxorubicin, SN-38 and rhodamine 123 Cai et al. (2010) TM3 Arg 482 Gly Arg 482 Thr (i) No change in the inhibitory activity of lapatinib; (ii) about two times greater inhibition by ritonavir, saquinavir and nalfinavir than in the WT variant; (iii) gaining the ability to transport rhodamine123 and doxorubicin; (iv) no influence on the transport of mitoxantrone; (v) loss of the ability to transport methotrexate Dai et al. (2008), Gupta et al. (2004), Honjo et al. (2001), Mitomo et al. (2003) Arg 482 Thr (i) Lower IC 50 of cyclosporine A for mutant than for WT variant; (ii) lower elacridar inhibition potency Xia et al. (2007) Arg 482 Lys Complete loss of transport activity Ejendal et al. (2006) Phe 489 Leu Impaired transport of porphyrins, no transport of methotrexate Tamura et al. (2006) Extracellular loop 3 Asn 590 Tyr Over twice reduced transport of mitoxantrone, topotecan, daunorubicin and rhodamine 123 Vethanayagam et al. (2005) Cys 592 Ala/Cys 608 Ala (i) Transport of mitoxantrone almost unchanged; (ii) transport of BODIPY-prazosin significantly impaired Henriksen et al. (2005a) Extracellular loop 3 Cys 603 Ser Cys 592 Ser/Cys 608 Ser Cys 592 Ser/Cys 603 Ser/Cys 608 Ser Diminished susceptibility to the inhibitory activity of fumitremorgin C Shigeta et al. (2010) Cys-less Arg 482 Gly-BCRP Complete loss of the ability to efflux mitoxantrone Liu et al. (2008b) The positions of the amino acid residues refer to the topological model of BCRP proposed by Wang et al. (2009). Login to comment