The relationship of autonomous homodimerization and signal activation to cell-survival capacity in the artificially generated TMD mutants of c-MPL. (A) A list of the AA substitution mutants generated at position Ser505 of c-MPL. All mutants were transfected into Ba/F3 cells. (B) MTT assay of the artificial TMD mutants of c-MPL in the absence of factor stimulation. Cells transfected with mutants with 2-polar residues (Glu, Asp, or Gln) showed factor-independent cell survival. Mock and Ba/F3 cells were transfected with PCI-Neo vector. The results are shown as means ± SEM of 3 separate experiments. (C) Autonomous homodimerization and signal activation of the artificial TMD mutants of c-MPL. The top panel shows the detection of homodimers in immunoblots under nonreducing conditions. BS³ was used as the cross-linker. ◀ indicate monomers (∼ 85 kDa) and dimers (∼ 170 kDa). The bottom panel depicts the phosphorylation status of MEK1/2 and STAT5 in the mutants of c-MPL with or without IL-3 stimulation. Only mutants carrying 2-polar residues (Glu, Asp, or Gln) showed autonomous homodimerization and phosphorylation of MEK1/2 and STAT5 in the absence of factor stimulation.