ABCG2 p.Ser187Ala
| Predicted by SNAP2: | A: D (66%), C: D (71%), D: D (80%), E: D (80%), F: D (85%), G: D (75%), H: D (80%), I: D (85%), K: D (85%), L: D (80%), M: D (80%), N: D (66%), P: D (85%), Q: D (75%), R: D (85%), T: D (63%), V: D (75%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, T: D, V: D, W: D, Y: D, |
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[hide] Functional characterization of human breast cancer... Mol Pharmacol. 2003 Dec;64(6):1452-62. Nakanishi T, Doyle LA, Hassel B, Wei Y, Bauer KS, Wu S, Pumplin DW, Fang HB, Ross DD
Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis.
Mol Pharmacol. 2003 Dec;64(6):1452-62., [PMID:14645676]
Abstract [show]
To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.
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No. Sentence Comment
4 Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity.
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ABCG2 p.Ser187Ala 14645676:4:120
status: VERIFIED57 The PCR product was ligated into the pCR Blunt TOPO II TABLE 1 Summary of cDNA constructs made as templates for producing BCRP cRNA Construct Plasmid Features of the BCRP cRNA Produced RNA Polymerase Consensus Sequence Upstream from BCRP cDNA I pSP64poly(A) R482T-poly(A) SP6 II pCR Blunt TOPO II Kozak-R482T-poly(A) T7 III pSD64TR 5ЈUTR-Kozak-R482T-3ЈUTR-poly(A) other BCRP forms: 5ЈUTR-Kozak-R482-3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/S187T- 3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/ S187A-3ЈUTR-poly(A) SP6 III- pSD64TR 5ЈUTR-R482T-3ЈUTR-poly(A) SP6 5ЈUTR, 3ЈUTR, portions of the 5Ј- and 3Ј-UTR of the Xenopus laevis beta-globin gene; Kozak, modified sequences proximate to the start codon, as described under Materials and Methods; Poly(A), addition of a poly(A) tail, as described under Materials and Methods.
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ABCG2 p.Ser187Ala 14645676:57:513
status: VERIFIED60 Introduction of S187T and S187A Mutations into Human BCRP cDNA.
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ABCG2 p.Ser187Ala 14645676:60:26
status: VERIFIED64 The S187T primer pair was 5Ј-G TTT ATC CGT GGT GTG AC T GGA GGA GAA AG-3Ј and 5Ј-CT TTC TCC TCC AGT CAC ACC ACG GAT AAA C-3Ј, and the S187A primer pair was 5Ј-G TTT ATC CGT GGT GTG GC T GGA GGA GAA AG-3Ј and 5Ј-CT TTC TCC TCC AGC CAC ACC ACG GAT AAA C-3Ј.
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ABCG2 p.Ser187Ala 14645676:64:158
status: VERIFIED163 To determine whether dimerization is required for BCRP activity, a mutant construct was created by substituting the highly conserved serine at residue 187 in the ABC signature motif with threonine (R482T/S187T) or alanine (R482T/S187A).
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ABCG2 p.Ser187Ala 14645676:163:229
status: VERIFIED199 Expression of dominant-negative construct was confirmed by Western blot (A) and by DNR accumulation (25 M, B) in control or R482T-expressing oocytes (f), R482T/S187T- expressing oocytes (o), or R482T/S187A-expressing oocytes (gray o).
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ABCG2 p.Ser187Ala 14645676:199:208
status: VERIFIED233 Hence, we made two mutations of serine 187 in this signature motif of BCRP R482T, one to an amino acid that, like serine, has a polar side chain (threonine, S187T) and the other to a nonpolar side chain amino acid (alanine, S187A).
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ABCG2 p.Ser187Ala 14645676:233:224
status: VERIFIED241 Although it is possible that the inhibition caused by the S187A/T mutants was the result of the mutant saturating a limiting and essential oocyte cellular component crucial for protein transport or maturation, it is more likely that the inhibition was analogous to a "dominant-negative" effect of the S187T mutant.
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ABCG2 p.Ser187Ala 14645676:241:58
status: VERIFIED[hide] ABCG2: the molecular mechanisms of urate secretion... Am J Physiol Renal Physiol. 2015 Sep 15;309(6):F485-8. doi: 10.1152/ajprenal.00242.2015. Epub 2015 Jul 1. Woodward OM
ABCG2: the molecular mechanisms of urate secretion and gout.
Am J Physiol Renal Physiol. 2015 Sep 15;309(6):F485-8. doi: 10.1152/ajprenal.00242.2015. Epub 2015 Jul 1., [PMID:26136557]
Abstract [show]
The human propensity for high levels of serum uric acid (SUA) is a trait that has defied explanation. Is it beneficial? Is it pathogenic? Its role in the human diseases like gout and kidney stones was discovered over a century ago [Richette P, Bardin T. Lancet 375: 318-328, 2010; Rivard C, Thomas J, Lanaspa MA, Johnson RJ. Rheumatology (Oxford) 52: 421-426, 2013], but today emerging new genetic and epidemiological techniques have revived an age-old debate over whether high uric acid levels (hyperuricemia) independently increase risk for diseases like hypertension and chronic kidney disease [Feig DI. J Clin Hypertens (Greenwich) 14: 346-352, 2012; Feig DI, Madero M, Jalal DI, Sanchez-Lozada LG, Johnson RJ. J Pediatr 162: 896-902, 2013; Feig DI, Soletsky B, Johnson RJ. JAMA 300: 924-932, 2008; Wang J, Qin T, Chen J, Li Y, Wang L, Huang H, Li J. PLoS One 9: e114259, 2014; Zhu P, Liu Y, Han L, Xu G, Ran JM. PLoS One 9: e100801, 2014]. Part of the mystery of the role uric acid plays in human health stems from our lack of understanding of how humans regulate uric acid homeostasis, an understanding that could shed light on the historic role of uric acid in human adaptation and its present role in human pathogenesis. This review will highlight the recent work to identify the first important human uric acid secretory transporter, ABCG2, and the identification of a common causal ABCG2 variant, Q141K, for hyperuricemia and gout.
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No. Sentence Comment
35 Using a Xenopus oocyte expression system, we demonstrated ABCG2 to be a high-capacity urate transporter with ABCG2-mediated c-14 uric acid efflux highly dependent on the intracellular concentration, and could be blocked by a specific ABCG2 inhibitor, FTC, or with a single amino acid substitution, S187A.
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ABCG2 p.Ser187Ala 26136557:35:298
status: NEW