Fig. 1.

Mutation of the TpoR W515 residue induces constitutive signaling via JAK2 and STAT5. (A) The indicated TpoR W515 mutants, where the W515 residue was replaced by aromatic residues (Left) or other residues (Right) were expressed in γ-2A cells cotransfected with JAK2 and tested for ligand-dependent and -independent induction of STAT5-dependent transcriptional activity. Two reporters are used in the dual luciferase assay: the experimental firefly (FF) luciferase reporter, which responds to JAK/STAT pathway, and a cotransfected renilla (RL) luciferase control reporter driven by a constitutive promoter that provides a baseline signal proportional to transfection levels. The firefly/renilla (FF/RL) ratio normalizes the FF response for experimental variability due to cell viability and transfection efficiency. (B) Constitutive activation of TpoR W515 mutants is reverted by substituting the preceding (R514) or subsequent (Q516) residue by tryptophan. Replacing residue 514 or 516 with a tryptophan residue (R514W or Q516W) reverts TpoR W515K (Left) and TpoR W515L (Right) to a wild-type TpoR phenotype. Shown in A and B are averages of three replicates ± SD in one representative experiment out of at least three. NS, nonsignificant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, Student t test.