The extracellular domain of TpoR, but not of EpoR, is indispensable for CALR mutants activity. (A) Schematic representation of engineered chimeric receptors. On the left, chimeric receptor TTE is composed of the extracellular and transmembrane domains of TpoR (in blue) fused to the cytosolic domain of EpoR (in red). On the right, chimeric receptor ETT is composed by the extracellular domain of EpoR (in red) fused to the transmembrane and cytosolic domains of TpoR (in blue). STAT5 transcriptional activity was measured by luciferase assay in γ2A cells transiently transfected with JAK2 and CALR wild-type or mutants in the presence of the chimeric receptor TTE (B) or ETT (C). Only the chimeric receptor presenting the extracellular domain of TpoR (TTE) is able to support constitutive activity induced by CALR mutants. Ba/F3 cells stably transduced with CALR constructs together with TTE (D) or ETT (E) were cultured for 3 days without cytokine to measure short-term autonomous growth using CellTiter-Glo technology. ETT cells were also tested for response to Epo, as positive control. (F) Long-term proliferation (day 10) of Ba/F3 expressing the indicated chimeric receptors and CALR variants. Values shown represent the average of 3 pooled independent experiments, each performed with 3 biological replicates ± SEM. Statistical analysis (jmp pro11) was performed by the nonparametric multiple comparisons Steel test with a control group; *P < .05, **P < .01, ***P < .001.