ABCMdb

ABCG2 p.Tyr645Phe

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Publications

PMID: 20739628 [PubMed] Ni Z et al: "Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport."

No. Sentence Comment

9 We substituted Asn387 , Gln398 , Asn629 , and Thr642 with Ala, Thr402 with Ala and Arg, and Tyr645 with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Login to comment
13 In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Login to comment
68 The forward primers used for mutagenesis were as follows: N387A (5=- AAG CGT TCA TTC AAA GCC TTG CTG GGT AAT CCC-3=), Q398A (5=-CAG GCC TCT ATA GCT GCG ATC ATT GTC ACA GTC-3=), T402A (5=-GCT CAG ATC ATT GTC GCA GTC GTA CTG GGA CTG-3=), N629A (5=-CTG GGG CTT GTG GAA GGC TCA CGT GGC CTT GGC TTG-3=), T642A (5=-GAT TGT TAT TTT CCT CGC AAT TGC CTA CCT GAA ATT G-3=), and Y645F (5=-TTC CTC ACA ATT GCC TTC CTG AAA TTG TTA TTT C-3=). Login to comment
158 To evaluate functional importance of these polar residues in substrate specificity and overall transport activity of BCRP, we generated six mutants in which Asn387 , Gln398 , Thr402 , Asn629 , and Thr642 were replaced with Ala. Tyr645 was replaced with Phe to assess the role of potential hydrogen bond of this residue in drug transport, but with the minimal probability of introducing structural changes. Login to comment
162 The levels of N387A, Q398A, T402A, N629A, T642A, and Y645F, determined by immunoblotting of whole cell lysates using beta-actin as an internal standard, were ϳ4.4-, 4.5-, 3.1-, 0.4-, 1.3-, and 1.5-fold that of wild-type BCRP (Fig. 2, A and B). Login to comment
189 After normalization to the BCRP protein levels, neither T642A nor Y645F seem to affect the efflux of MX and BODIPY-prazosin compared with wild-type protein, except for a moderate decrease in the efflux of Hoechst 33342. Login to comment
200 Representative areas of HEK-293 cells expressing wild-type BCRP and the mutants N387A, Q398A, T402A, N629A, T642A, and Y645F are shown. Login to comment
224 The Km values of N387A, T402A, T642A, and Y645F were comparable to that of wild-type protein; however, the Km values of Q398A and N629A were decreased by ϳ50-60%. Login to comment
229 The Vmax/Km values of Q398A, T642A, and Y645F were comparable to that of wild-type protein. Login to comment
231 FTC-inhibitable efflux activities of HEK-293 cells stably expressing wild-type and mutant BCRP Mitoxantrone BODIPY-Prazosin Hoechst 33342 ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio Wild-type BCRP 10.6 Ϯ 0.6 10.6 Ϯ 0.6 1.0 12.5 Ϯ 4.3 12.5 Ϯ 4.3 1.0 2150.0 Ϯ 25.5 2150.0 Ϯ 25.5 1.0 N387A 11.3 Ϯ 0.5 2.5 Ϯ 0.1* 0.2 54.5 Ϯ 5.8 12.3 Ϯ 1.3 1.0 5,024.7 Ϯ 570.5 1,131.7 Ϯ 128.5* 0.5 Q398A 28.1 Ϯ 5.2 6.3 Ϯ 1.2* 0.6 69.3 Ϯ 10.6 15.5 Ϯ 2.4 1.2 4,206.7 Ϯ 252.1 941.1 Ϯ 56.4* 0.4 T402A 12.3 Ϯ 2.4 4.0 Ϯ 0.8* 0.4 4.5 Ϯ 1.8 1.5 Ϯ 0.6* 0.1 999.3 Ϯ 352.9 322.4 Ϯ 113.8* 0.1 N629A 19.3 Ϯ 3.7 46.0 Ϯ 8.8* 4.3 12.8 Ϯ 1.5 30.5 Ϯ 3.6* 2.4 2,681.0 Ϯ 370.5 6,383.3 Ϯ 882.1* 3.0 T642A 17.0 Ϯ 0.3 12.9 Ϯ 0.2 1.2 15.4 Ϯ 1.3 11.8 Ϯ 1.0 0.9 1,860.7 Ϯ 462.3 1,420.4 Ϯ 352.9* 0.7 Y645F 16.8 Ϯ 2.3 11.6 Ϯ 1.6 1.1 15.1 Ϯ 4.6 10.4 Ϯ 3.2 0.8 1,358.7 Ϯ 35.5 937.0 Ϯ 24.5* 0.4 Values are means Ϯ SD of 3 independent experiments. Login to comment
238 Relative drug resistance of HEK-293 cells expressing wild-type and mutant BCRP MX SN-38 Dox Rho-123 IC50, nM RR (ratio) IC50, nM RR (ratio) IC50, nM RR IC50, nM RR pcDNA control 8.5 Ϯ 0.6 2.3 Ϯ 0.28 125.6 Ϯ 32.0 1,881 Ϯ 168.2 Wild-type BCRP 64.8 Ϯ 3.9 7.6 (1.0) 89.7 Ϯ 5.4 39.0 (1.0) 168.0 Ϯ 24.5 1.3 3,341 Ϯ 267.4 1.8 N387A 54.8 Ϯ 6.3 6.4 (0.2)* 44.8 Ϯ 6.3 19.5 (0.1)* 120.4 Ϯ 31.6 1.0 3,279 Ϯ 436.8 1.7 Q398A 49.9 Ϯ 4.6 5.9 (0.2)* 71.6 Ϯ 4.3 31.1 (0.2)* 173.4 Ϯ 36.5 1.4 2,534 Ϯ 376.5 1.3 T402A 43.3 Ϯ 7.6 5.1 (0.2)* 63.0 Ϯ 5.4 27.4 (0.2)* 129.4 Ϯ 31.4 1.0 2,140 Ϯ 210.7 1.1 N629A 76.5 Ϯ 12.3 9.0 (2.8)* 108.3 Ϯ 12.3 47.1 (2.9)* 164.6 Ϯ 14.8 1.3 3,051 Ϯ 286.5 1.6 T642A 50.0 Ϯ 9.5 5.9 (0.6) 49.9 Ϯ 9.9 21.7 (0.4)* 156.1 Ϯ 20.0 1.2 1,393 Ϯ 221.6 0.7 Y645F 42.8 Ϯ 6.8 5.0 (0.5)* 44.8 Ϯ 6.3 19.5 (0.3)* 132.4 Ϯ 18.4 1.1 1,846 Ϯ 206.2 1.0 The IC50 values shown are means Ϯ SD of 3 independent experiments. Login to comment
250 Only a slight decrease in 5D3-phycoerythrin fluorescence was observed for the vector control cells (Fig. 5); however, the 5D3-phycoerythrin fluorescence was differentially increased in a prazosin concentration-dependent manner for wild-type BCRP, N629A, T642A, and Y645F. Login to comment
274 Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP Wild-type BCRP N387A Q398A T402A N629A T642A Y645F Vmax, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 16.9 Ϯ 1.5 14.9 Ϯ 0.93 17.4 Ϯ 1.7 11.5 Ϯ 1.1 17.9 Ϯ 1.9 14.5 Ϯ 1.9 Vmax normalized to BCRP protein level, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 7.0 Ϯ 0.63 6.5 Ϯ 0.41 8.3 Ϯ 0.81 28.0 Ϯ 2.7 16.3 Ϯ 1.7 12.0 Ϯ 1.6 Km for ATP, mM 0.89 Ϯ 0.40 0.90 Ϯ 0.27 0.48 Ϯ 0.12 1.1 Ϯ 0.35 0.40 Ϯ 0.15 0.93 Ϯ 0.34 0.89 Ϯ 0.43 Vmax/Km, nmol Pi ·min-1 ·mg protein-1 ·mM-1 16.3 7.8 13.5 7.5 70.0 17.5 13.5 Values are means Ϯ SD of 3 independent determinations. Login to comment
339 A conservative mutation of Tyr645 to Phe and Ala substitution of Thr642 caused only moderate changes in drug efflux and resistance that were restricted to Hoechst 33342 and SN-38 (Tables 1 and 2). Login to comment
350 Prazosin differentially increased 5D3 binding to wild-type BCRP, N629A, T642A, and Y645F, whereas 5D3 binding to N387A and Q398A was slightly decreased (Fig. 5). Login to comment

PMID: 25445676 [PubMed] Gal Z et al: "Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter."

No. Sentence Comment

5 We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. Login to comment
114 We found that the Y469F and Y645F mutants were active (Fig. 2B), indicating the importance of the phenyl ring, but not of a hydroxyl group at these positions for protein function. Login to comment
120 We found that the basal ATPase activity of the Y469F, Y570S and Y645F mutants showed a moderate (approximately 20%, p b 0.05) increase upon cholesterol addition, while the substrate stimulated ATP hydrolysis of the same mutants was significantly (50-100% increase, p b 0.01) accelerated by cholesterol loading (Fig. 2C). Login to comment
174 generated mammalian HEK 293 cells stably expressing the Y413S, Y469F, Y570S and Y645F mutants. Login to comment
233 Previously, the Y645F mutation has been found to be functional, although has shown slightly decreased Hoechst 33342 transport capacity [39]. Login to comment
250 BODIPY-prazosin Pheophorbide A Hoechst 33342 mitoxantrone wtABCG2 + + + + Y413S + + + + Y469F + + + + Y570S + + + + Y645F + + + + Fig. 8. Login to comment