Article Figures & Data
Figures
- Figure 1.
Protein expression and purification. A, domain 11 of human IGF2R was cloned into the expression vectors pPIC9K and pMT/BiP/V5-His B; the latter vector, including a COOH-terminal human IgG1 Fc tag for dimerization. B, mutations were introduced at IGF2R residues 1544 and 1572. Proteins 11-Fc, 11E1544K-Fc, and 11I1572A-Fc were expressed in Drosophila D.Mel-2 cells and purified via protein A-Sepharose affinity chromatography. The single domain proteins, WT domain 11, 11E1544K, and 11I1572A were expressed in P. pastoris and purified via nickel affinity chromatography. Aliquots of eluate (10 μL) were electrophoresed on 12% SDS-PAGE under reducing conditions and Coomassie stained. C, Fc-tagged proteins were further purified under nondenaturing conditions via a gel filtration column calibrated against protein standards. Representative A280 nm elution trace for 11-Fc.
- Figure 2.
Binding of Fc-tagged and untagged domain 11 proteins to IGF-II. A, non-Fc-tagged proteins were injected over an IGF-II–coated BIAcore SA sensor chip flow cell surface at a flow rate of 40 μL min−1 and a range of concentrations (32, 64, 128, 256, 512, and 2048 nmol/L). Association was for 2 min and dissociation was for 4 min. Data from buffer controls and a reference flow cell were subtracted and data were fitted to a two-state conformational change model using BIAevaluation software. Data were from Zaccheo et al.(44). Representative fitted sensorgrams for WT domain 11 (11WT; left) and domain 11E1544K (right). B, Fc-tagged proteins were injected over the same chip at a flow rate of 75 μL min−1 and a different range of concentrations (2.464, 1.232, 0.616, 0.308, 0.154, and 0.077 nmol/L). Association was for 3 min and dissociation was for 60 min. Data from buffer controls and a reference flow cell were subtracted and data were fitted to a bivalent analyte model using BIAevaluation software. Representative fitted sensorgrams for 11WT-Fc (top) and 11E1544K-Fc (bottom). C and D, control for mass transfer limitation. Protein 11E1544K-Fc was injected over an IGF-II–coated BIAcore SA sensor chip flow cell surface at 0.616 nmol/L at a range of flow rates (5, 20, 40, 60, and 75 μL min−1). Association was for 3 min and dissociation was for 60 min. Data were aligned using BIAevaluation software. C, aligned sensorgrams of the data. D, graph of flow rate versus amplitude of binding.
- Figure 3.
IGF-II dose-dependent increase in phosphorylation of PKB and cell proliferation. A, HaCaT; B, MEF. Increasing doses of IGF-II lead to an increase in phosphorylation of PKB in immortalized MEFs homozygous null for Igf2 (Igf2−/−) and HaCaT cell lines. Samples were probed with an anti–phospho-(Ser473) PKB antibody. Equal loading was confirmed by reprobing the blot with an antibody to α-tubulin. Increasing doses of IGF-II lead to an increase in cell proliferation as measured by [3H]thymidine incorporation in HaCaT cells (C) and Igf2−/− MEFs (D). The experiment was done at least thrice with each dose done in triplicate. Points, mean; bars, SE.
- Figure 4.
Inhibition of IGF-II–induced signaling and cell proliferation by domain 11 ligand traps. Domain 11 constructs with two different mutations, E1544K (which enhances IGF-II binding) and I1572A (which inhibits IGF-II binding), either with or without an Fc tag, were compared in their ability to block the actions of IGF-II in Igf2−/− MEFs. A, Igf2−/− MEFs were stimulated for 24 h with 6.5 nmol/L IGF-II preincubated for 10 min at room temperature with the domain 11 constructs indicated at 650 or 1,300 nmol/L (100-fold and 200-fold molar ratio, respectively). At 1,300 nmol/L, 11E1544K had the equivalent number of IGF-II binding sites as 650 nmol/L 11E1544K-Fc. However, only the homodimer, 11E1544K-Fc, significantly decreased [3H]thymidine incorporation compared with IGF-II alone. Columns, average (n = 3); bars, SE. *, P = 0.04. B, IGF-II–stimulated phosphorylation of PKB and MAPK was inhibited by preincubation with 11E1544K-Fc only. Again, the domain 11 monomers were unable to inhibit the actions of IGF-II even with equivalent numbers of IGF-II binding sites present. Equal loading was confirmed by reprobing the blot with an anti–α-tubulin antibody. C, comparison of Fc homodimer proteins. The 11E1544K-Fc construct significantly inhibited IGF-II–stimulated proliferation compared with 11-Fc, although the affinity for IGF-II is the same order of magnitude (see Table 2). Columns, mean (n = 3 experiments with triplicate samples); bars, SE. *, P = 0.024.
- Figure 5.
11E1544K-Fc decreases IGF-II–stimulated proliferation and signaling in a dose- and IGF-II–dependent manner. A, HaCaT cells were stimulated with 6.5 nmol/L IGF-II and increasing doses of 11E1544K-Fc. A significant decrease in [3H]thymidine incorporation was seen with 650 nmol/L (P = 0.005) and 1,300 nmol/L (P = 0.013) 11E1544K-Fc. Points, average (n = 3 experiments with triplicate samples); bars, SE. Inset, the decrease in [3H]thymidine uptake was equated to nanomol per liter of active IGF-II remaining. Six hundred fifty and 1,300 nmol/L IGF2R11E1544K-Fc reduced proliferation by 50% and 73%, respectively, and the respective amount of active IGF-II was reduced by 82% and 90%. B, cells were stimulated with 6.5 nmol/L IGF-II or IGF-I and 650 nmol/L 11E1544K-Fc or 11I1572A-Fc. 11I1572A-Fc failed to inhibit IGF-II–stimulated [3H]thymidine incorporation in both HaCaT and immortalized Igf2−/− MEFs. IGF-I, which also stimulates [3H]thymidine incorporation in HaCaT cells, was not inhibited by addition of 650 nmol/L 11E1544K-Fc. 11E1544K-Fc significantly inhibited IGF-II–stimulated proliferation in HaCaT cells (**, P = 0.005) and Igf2−/− MEFs (*, P = 0.034). Columns, average (n = 6 experiments with samples in triplicate); bars, SE. C, 11E1544K-Fc blocks IGF-II–stimulated activation via the IGF1R. Cells were stimulated with 1.3 nmol/L IGF-II or IGF-I and 130 nmol/L 11E1544K-Fc or 11I1572A-Fc. 11E1544K-Fc inhibited IGF-II–stimulated but not IGF-I–stimulated phosphorylation of PKB in HaCaT and Igf2−/− MEFs. 11I1572A-Fc failed to inhibit IGF-II–stimulated phosphorylation. Equal loading was confirmed by reprobing the blots with an antibody to α-tubulin.
Tables
- Table 1.
Calculated masses, isoelectric points, and molar extinction coefficients of the proteins studied
Protein Molecular weight (kDa) pI Extinction coefficient (mol/L−1 cm−1 at 280 mm) 11wild-type 16.82 7.76 13,650 11E1544K 16.82 8.45 13,650 11I1572A 16.77 7.76 13,650 11wild-type-Fc 88.06 8.02 94,420 11E1544K 88.06 8.36 94,420 11I1572A 87.98 8.02 94,420 -
Abbreviation: pI, isoelectric point.
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- Table 2.
Kinetic and affinity constants for IGF-II binding to IGF2R domain 11 proteins studied
Protein Major kinetic variables Minor kinetic variables kal (×105 mol/L−1 s−1) kdl (×10−2 s−1) KD (×10−9 mol/L) ka2 (×10−3 s−1) ka2 (×10−5 RU−1 s−1) kd2 (×10−4 s−1) 11wild-type 6.62 ± 0.13 7.87 ± 0.29 118.8 ± 3.5 2.45 ± 0.33 — 121 ± 7.2 11E1544K 20.23 ± 2.97 4.06 ± 0.28 20.5 ± 2.0 3.83 ± 1.10 — 79 ± 28 11I1572A — — — — — — 11wild-type-Fc 13.65 ± 0.01 0.445 ± 0.04 3.26 ± 0.3 — 7.12 ± 0.19 2.33 ± 0.12 11E1544K 22.27 ± 0.74 0.401 ± 0.03 1.79 ± 0.08 — 7.86 ± 0.04 1.26 ± 0.03 11I1572A — — — — — — -
NOTE: The values given correspond to the average value ± the SE of three independent experiments. We have published the kinetic constants for non-Fc-tagged domain 11 proteins previously (44).
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Abbreviation: RU, resonance unit.
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Volume 6, Issue 2
