Lower MPL P106L activity is related to the cell-surface receptor number. (A) MPL receptor internalization after THPO binding: receptor cell-surface expression in UT-7 MPLWT and UT-7 MPL P106L clone 1 was measured by flow cytometry at different times after THPO binding. Analyzed were 2 × 10⁵ cells per point, and results are means ± standard deviation of 3 independent experiments. (B) MPL binding to [¹²⁵I]THPO (upper panel): the specific radioactivity bound to 5 × 10⁵ UT-7 parental cells (empty circle), MPL WT (filled circle), P106L (empty square), and P106L clone 1 (filled square) UT-7 cells following 2-hours incubation at 15°C with radiolabeled ligand. A representative of 3 independent experiments is shown, together with a nonlinear fit to a 1-site model of specific saturation binding for UT-7 MPL WT and UT-7 MPL P106L clone 1. Binding to UT-7 parental and UT-7 MPL P106L cells was too low in this assay to unambiguously fit to model. Scatchard plot of [¹²⁵I]THPO binding to UT-7 MPL WT and P106L cells (lower panel). (C) UT-7 MPL transduced cell proliferation in response to eltrombopag: UT-7 MPL WT, P106L, and THPO-selected clones were washed and then cultivated with THPO or eltrombopag at a concentration of 1 × 10⁵ cells/mL and compared with the control GM-CSF, representing 100%. Viable cells were counted using KOVA slide. (D) UT-7 MPL WT and UT-7 MPL P106L signaling induced by THPO and eltrombopag. Cells were starved of cytokine for 5 hours and then stimulated with various concentrations of THPO or eltrombopag and compared with the control (GM-CSF). STAT1, STAT3, STAT5, AKT, and ERK1/2 activation was analyzed by western blotting using corresponding antibodies. Cell-surface MPL expression data for UT-7 MPL WT and MPL P106L clone 1 correspond to Figure 3A.