Derivation and validation of hyperactive CGS variants. (A) Schematic comparing the principal components of the Tpo receptor Mpl and the hybrid Mpl-based CGS receptor. Tpo and CID not to scale. (B) Schematic indicating the positions of the amino acid substitutions introduced into the Mpl signaling domain of the CGS receptor. (C) Design of the lentiviral vectors expressing the CGS variants Y* (Y582F), K*K* (K544F and K564F), and K*K*Y*. Coordinates are based on amino acid sequence of mouse Mpl. (D) Hyperactive variants improve CGS-mediated expansion of Ba/F3 cells. Murine Ba/F3 cells were transduced with lentivectors expressing the CGS containing the wt, Y*, K*K*, or K*K*Y* Mpl signaling domains. After confirming similar rates of gene transfer, cells were cultured in the indicated concentration of the CID AP1903. After 5 days, cell proliferation was assessed by the MTS assay. Data represent the mean ± SD of triplicate determinations. P values are based on the t test across all concentrations. (E) The hyperactive variant Y* improves CGS receptor stability. Ba/F3 cells transduced with the wt or Y* CID lentivectors and expanded in 100 nM CID AP20187 were treated with CYH for 4 and 8 hours, and the level of wt and Y* CID receptor proteins was compared with untreated controls (0 μg/mL CYH) by western blotting using an HA tag antibody. The relative levels of CGS protein compared with the untreated controls are shown at the bottom. ΔLTR, self-inactivating long-terminal repeat; CYH, cycloheximide; GFP, GFP reporter gene; MSCVpro, MSCV promoter; PGKpro, PGK gene promoter; SD, standard deviation.