Receptor cell surface localization is required for complete transforming activity of CALR mutants in Ba/F3 cells. (A) Schematic representation of TpoR P106L and TpoR Cys less showing the mutated residues. The activating effect of CALR mutants on a series of mutated receptors measured by luciferase assay (B), short-term proliferation assay (C, upper graph) and long-term proliferation assay (C, under graph), and the cell surface localization of the receptors measured by flow cytometry (D). Values shown in the luciferase assay and short-term proliferation assay represent the average of 3 independent experiments each done with 3 biological repeats ± standard error of the mean, and values shown in the long-term proliferation assay correspond to 3 replicates ± standard error of the mean. Statistical analysis (jmp pro12) was performed by using the nonparametric multiple comparisons Steel test with a control group. (E) Cartoon showing mechanisms of action for CALR mutants requiring binding to TpoR through Asn117 and a hydrophobic patch near the Asn117 site (left cartoon). The oncogenic effect of CALR mutants requires binding and stabilization of the receptor, exit from the ER, and traffic through the secretory pathway. Intracellular TpoR/CALR mutant complexes can induce an incomplete early signal (right cartoon, 1). Only cell surface localization of this complex leads to complete activation and cell transformation (right cartoon, 2).