Noncysteine mutations in IL7R and CRLF2 cause constitutive activation of the JAK-STAT pathway and increase receptor dimerization. (A) Constitutive phosphorylation of Stat5 in BaF3 cells expressing IL7R mutants after 5 hours of cytokine deprivation. One mutant with insertion of cysteine (IL7R^insPPCL) was used as a positive control. IL-3+ indicates cells harvested after 5 hours of IL-3 deprivation followed by 20 minutes of IL-3 stimulation. (B) Identification of constitutive phosphorylation of Jak1, Jak2, and Stat3 in BaF3 cells expressing IL7R mutants after 5 hours of cytokine deprivation. IL-3+ indicates cells harvested after 5 hours of IL-3 deprivation followed by 20 minutes of IL-3 stimulation. Cells were subjected to lysis and immunoprecipitation with anti p-Tyr antibody (sc-508; Santa Cruz Biotechnology). The presence of Jak1, Jak2, and Stat3 was visualized by western blotting with anti-Jak1/Jak2/Stat3 antibodies. (C) Constitutive phosphorylation of Stat5 in BaF3 cells expressing CRLF2 mutant after 5 hours of cytokine deprivation. IL-3+ indicates cells harvested after 5 hours of IL-3 deprivation followed by 20 minutes of IL-3 stimulation. (D) Identification of constitutive phosphorylation of Jak2 and Stat5 in BaF3 cells expressing CRLF2 mutants after 5 hours of cytokine deprivation. IL-3+ indicates cells harvested after 5 hours of IL-3 deprivation followed by 20 minutes of IL-3 stimulation. Cells were subjected to lysis and immunoprecipitation with anti p-Tyr antibody (sc-508; Santa Cruz Biotechnology). The presence of Jak2 and Stat5 was visualized by western blotting with anti-Jak2 or Stat5 antibodies. (E) Relative dimerization level of IL7R and CRLF2 from HEK293 cells transiently transfected with the WT or mutant receptor. Dimerization was calculated by dividing luminescence by the mean fluorescence intensity of each treatment, normalizing the luminescence signal for experimental variability resulting from transfection efficiency. * P < .01, 1-way ANOVA, and Student t test. (F) Alignment of WT, natural mutant (EKV), and 6 experimental mutants (numbered 1 to 6) in the TMD of IL7R. Numbers show the positions of nucleotides and corresponding amino acids. The inserted nucleotides and amino acids are shown in red. The deleted AL amino acids are shown in green. (G) Cytokine withdrawal assay of BaF3 cells transduced with IL7R experimental mutants. (H) Constitutive phosphorylation of Stat5 in BaF3 cells expressing IL7R experimental mutants after 5 hours of cytokine deprivation. IL-3+ indicates cells harvested after 5 hours of IL-3 deprivation followed by 20 minutes of IL-3 stimulation. Del, deletion; Ins, insertion.