Article Figures & Data
Figures
- Fig. 1.
Determination of the differential 3′-deoxy-nucleoside analog (AZT and DDI) transport by hENTs. (A) Transport activities of 3H-AZT (20 µM) were measured and plotted at pH 7.4 of oocytes at 25°C 24 hours after injection of H2O, hENT1, or hENT2 transcripts. (B) Transport activities of 3H-AZT (20 µM) at pH 5.5 after injection of oocytes with H2O or ∆36hENT3 transcripts. (C) Transport activities of 3H-DDI (20 µM) were measured and plotted at pH 7.4 of oocytes at 37°C 24 hours after injection of H2O, hENT1, or hENT2 transcripts. (D) Transport activities of 3H-DDI at pH 5.5, with H2O or ∆36hENT3 transcript injection. ****P < 0.0001 (one-way analysis of variance/t test with hENTs compared with H2O). Bars represent the average ± S.E. (n = 8–12 oocytes).
- Fig. 2.
Generation and transport characterization of chimeras of hENT3 and hENT1. (A) Diagrammatic representation of the hENT3 and hENT1 chimeras constructed by replacement of the C-terminal hENT3 region by corresponding regions of hENT1. N-terminal segments of hENT3 and C-terminal segments of hENT1 were joined with increasing increments of approximately 40 amino acids from ∆36hENT3. The constructs were named as in the following example: 80(3+1) is 80 amino acids starting from the N terminus of hENT3 and the remaining length of the protein from hENT1. (B) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C and D) Uptake of 3H-adenosine (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts in the absence and presence of NBMPR at pH 5.5 (C) and pH 7.4 (D), respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
- Fig. 3.
Identification of the region(s) of hENT3 responsible for 3′-deoxy-nucleoside analog transport by hENT1 and hENT3 chimeras. (A) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (B) Uptake of 3H-AZT (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C) Uptake of 3H-DDI (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, and chimeric transcripts. *P < 0.05; **P < 0.01; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
- Fig. 4.
Determination of relative dominance of the intracellular targeting signal(s) of N-terminal 36 amino acids of hENT3 and the C-terminal half of hENT1 in deciding cell surface transport activity. (A) Diagrammatic representation of the 267(3+1) chimera with or without the N-terminal 36 amino acids from hENT3. (B) Uptake of 3H-adenosine (20 µM) into oocytes was measured and plotted at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (C) Uptake of 3H-AZT (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. (D) Uptake of 3H-DDI (20 µM) into oocytes at 25°C 24 hours after injection of hENT1, ∆36ENT3, or chimeric transcripts. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance). Data represent the average ± S.E.M. (n = 8–12 oocytes).
- Fig. 5.
Identification of residues dictating the selectivity of hENT3 in transporting 3′-deoxy-nucleoside analogs. (A) Sequence alignment of hENT1, hENT2, and hENT3: the residues that are common in hENT2 and hENT3 but not in hENT1 are indicated in the boxes. (B–D) Transport activities of 3H-adenosine (20 µM; B), 3H-AZT (20 µM; C), and 3H-DDI (20 µM; D) of H2O, ∆36hENT3, and mutant transcript-injected oocytes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way analysis of variance of mutants compared with ∆36hENT3). For (C) and (D), statistical analysis was performed for mutants retaining adenosine transport activity. Data represent the average ± S.E.M. (n = 8–12 oocytes).
Tables
Construct Forward Primer Reverse Primer hENT1 5′-GATTA GTCGAC CCACC ATG ACA ACC AGT CAC CAG-3′ 5′-GTA TCTAGA TCA CAC AAT TGC CCG-3′ hENT2 5′-TTC AAGCTT CCACC ATGGCGCGAGGAGACG-3′ 5′-GTA TCTAGA TCAGAGCAGCGCCTTGAAGA-3′ hENT3 chimera 80(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-GGA CAT GTC CAG GCG GAA CAT CCA GTA CTC-3′ 120(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-CAG GAA GGA GTT GAG CAC CAG GCA CAG CAT-3′ 161(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-GAC AAA GAA GGG CAG GGT CCA GGA GGA AGT-3′ 200(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-CTG GCC ACT CAT GAT TGC CTG GGA GTT CCT-3′ 240(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-GAT GGT CAA AAT GAT GAA GAC AGT GGC CGT-3′ 267(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-CAA CTT GGT CTC CTG CTC CCC GGC CGC AAG AAC-3′ Full-length 267(3+1) 5′-GATTA GTCGAC CCACC ATG GCC GTT GTC TCA GAG GAC-3′ 5′-CAA CTT GGT CTC CTG CTC CCC GGC CGC AAG AAC-3′ 280(3+1) 5′-GATTA GTCGAC CCACC ATG GCC GTT GTC TCA GAG GAC-3′ 5′-TGA AAC TCC AGA TTC GGA GTC CTG GGG AAG-3′ 320(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-GGC TGG AAA CAT CCC GCT GGT GAT GAA GAA-3′ 360(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-GAG GCT CCG GCC CAA TAG GTC AGC AAA GTT-3′ 400(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-AGT CAG GTA GCG GCG GGG CTG GTA GTT ACA-3′ 440(3+1) 5′-GATTA GTCGAC CCACC ATG GAC CGC CCG CCC CCT GGCC-3′ 5′-CTC AGC TGG CTT CAC AAT CTT AGG CCC GTA-3′ hENT1 chimera 80(3+1) 5′-GAG TAC TGG ATG TTC CGC CTG GAC ATG TCC-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 120(3+1) 5′-CAG GAA GGA GTT GAG CAC CAG GCA CAG CAT-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 161(3+1) 5′-ACT TCC TCC TGG ACC CTG CCC TTC TTT GTC-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 200(3+1) 5′-AGG AAC TCC CAG GCA ATC ATG AGT GGC CAG-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 240(3+1) 5′-ACG GCC ACT GTC TTC ATC ATT TTG ACC ATC-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 267(3+1) 5′-GTT CTT GCG GCC GGG GAG CAG GAG ACC AAG TTG-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ Full-length 267(3+1) 5′-GTT CTT GCG GCC GGG GAG CAG GAG ACC AAG TTG-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 280(3+1) 5′-CTT CCC CAG GAC TCC GAA TCT GGA GTT TCA-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 320(3+1) 5′-TTC TTC ATC ACC AGC GGG ATG TTT CCA GCC-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 360(3+1) 5′-AAC TTT GCT GAC CTA TTG GGC CGG AGC CTC-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 400(3+1) 5′-TGT AAC TAC CAG CCC CGC CGC TAC CTG ACT-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ 440(3+1) 5′-TAC GGG CCT AAG ATT GTG AAG CCA GCT GAG-3′ 5′-GTA ACTAGT TCA CAC AAT TGC CCG-3′ Mutant 55I>L 5′-TGT GGC ACA TAC TTA ATC TTC TTC AGC-3′ 5′-GCTGAAGAAGATTAAGTATGTGCCACA-3′ 72I>M 5′-TGG AAC TTC TTT ATG ACT GCC AAG GAG-3′ 5′-CTCCTTGGCAGTCATAAAGAAGTTCCA-3′ 131V>I 5′-AAC AGG GTT GCA ATC CAC ATC CGT GTC-3′ 5′-GACACGGATGTGGATTGCAACCCTGTT-3′ 156D>Q 5′-CTG GTG AAG GTG CAG ACT TCC TCC TGG-3′ 5′-CCAGGAGGAAGTCTGCACCTTCACCAG-3′ 158S>D 5′-AAG GTG GAC ACT GAG TCC TGG ACC CGT-3′ 5′-ACGGGTCCAGGACTCAGTGTCCACCTT-3′ 179S>G 5′-CTC AGC GGT GCC GGT ACT GTC TTC AGC-3′ 5′-GCTGAAGACAGTACCGGCACCGCTGAG-3′ 181V>I 5′-GGT GCC TCC ACT ATT TTC AGC AGC AGC-3′ 5′-GCTGCTGCTGAAAATAGTGGAGGCACC-3′ 198S>T 5′-CCT ATG AGG AAC ACT CAG GCA CTG ATA-3′ 5′-TATCAGTGCCTGAGTGTTCCTCATAGG-3′ 213A>S 5′-GGG ACG GTC AGC TCC GTG GCC TCA TTG GTG-3′ 5′-CACCAATGAGGCCACGGAGCTGACCGTCCC-3′ 225D>E 5′-GCT GCA TCC AGT GAG GTG AGG AAC AGC-3′ 5′-GCTGTTCCTCACCTCACTGGATGCAGC-3′ 231L>F 5′-AGG AAC AGC GCC TTT GCC TTC TTC CTG ACG-3′ 5′-CGTCAGGAAGAAGGCAAAGGCGCTGTTCCT-3′ 239V>A 5′-CTG ACG GCC ACT GCC TTC CTC GTG CTC-3′ 5′-GAGCACGAGGAAGGCAGTGGCCGTCAG-3′
Data Supplement
- Supplemental Figure -
Supplemental Figure 1 - Spice site positions of hENT3-hENT1 chimeras
- Supplemental Figure -
