ABCMdb

ABCG2 p.Ser187Thr

None has been submitted yet.

Publications

PMID: 14645676 [PubMed] Nakanishi T et al: "Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis."

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. Login to comment
5 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. Login to comment
57 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. Login to comment
60 Introduction of S187T and S187A Mutations into Human BCRP cDNA. Login to comment
64 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. Login to comment
163 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). Login to comment
181 Indeed, when BCRP (R482T) was coinjected with varying amounts of the "dominant-negative" construct S187T/R482T, the transport of DNR was significantly inhibited in a manner dependent on the amount of the S187T mutant construct added (Fig. 5C), strongly suggesting that homodimerization or multimerization is essential for BCRP activity. Login to comment
182 The degree of inhibition seemed to plateau after the addition of 48 ng of the "dominant-negative" construct; for technical reasons, we were unable to inject the oocytes with amounts of S187T mutant cRNA in excess of 72 ng. Login to comment
199 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). Login to comment
201 DNR accumulation (25 ␮M, C) was determined in oocytes coinjected with 24 ng of R482T cRNA and different amounts (0 to 72 ng) of the R482T/S187T construct cRNA (f). Login to comment
202 As control, the accumulation of 25 ␮M DNR was also determined in control (Ⅺ) or oocytes injected with 24 ng of R482T/S187T cRNA only (o). Login to comment
233 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). Login to comment
239 When the S187T mutation was coexpressed with BCRP R482T in the oocytes, the S187T mutant protein acted as would a dominant-negative inhibitor of BCRP transport of DNR, with the degree of inhibition increasing in proportion to the amount of S187T cRNA injected. Login to comment
241 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. Login to comment
245 This may have occurred because the affinity of A-to-A and I-to-I homodimeric partners may be greater than that of the S187T mutant-to-active BCRP heterodimeric partners. Login to comment
247 Because of injection volume constraints and the viscosity of highly concentrated cRNA solutions, we were technically unable to inject the oocytes with amounts of the S187T mutant form in excess of 72 ng. Login to comment

PMID: 19506252 [PubMed] Woodward OM et al: "Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout."

No. Sentence Comment

69 The physiological importance of ABCG2 in humans is illustrated by 0.0 0.5 1.0 1.5 noitalumuccAetarU nim021/etycoo/lomp H2O WT WT +FTC S187T 0 100 200 300 400 0 2 4 noitalumuccAetarU nim06/etycoo/lomp Urate Concentration ( µM) 0 2 4 6 8 10 0.00 0.05 0.10 nim/etycoo/lomp:xulffE Internal oocyte concentration (µM) 0 20 40 60 0.4 0.6 0.8 1.0 gniniameretarUevitaleR * 0.0 0.5 1.0 1.5 noitalumuccAetarU nim021/etycoo/lomp H2O MRP4ABCG2 ** A B C D E ** ** 500 ** ** ** ** * ** ** ** G F Time (min) 0.0 0.5 1.0 1.5 2.0 eAccumulationtarUevitaleR ** LLCPK : control +FTC basal brush border membrane Fig. 1. Login to comment
72 (B) Urate accumulation in oocytes injected with H2O, ABCG2 WT mRNA (incubated with or without 5 ␮M FTC), or the nonfunctional ABCG2 mutant S187T (N ϭ 10 samples each and n ϭ 20 oocytes each for all accumulation experiments). Login to comment
73 (C) Urate accumulation is dependent on the extracellular urate concentration (H2O [blue circles]; S187T [black squares]; WT [red triangles]; N ϭ 10 and n ϭ 20 each). Login to comment