TpoR constitutive activation induced by CALR mutants is abrogated by the TpoR soluble form and requires the glycan binding site of CALR. (A) The soluble extracellular domain of TpoR acts as a competitive inhibitor of TpoR activation by the CALR mutants as shown by luciferase assay (STAT5 transcriptional activity). This inhibitory effect is lost when all the N-glycosylated asparagine residues of the soluble TpoR are mutated to glutamine (soluble TpoR-NtQ) (B). (C) The γ2A luciferase assay showed that the TpoR -D261A/L265A mutant, unable to bind Tpo, is activated by CALR del52. CALR del52 mutant unable to bind N-glycosylation modifications (CALR del52 D135L/Y109F) do not induce TpoR -dependent STAT5 activation (D). Values shown represent the average of 3 pooled independent experiments, each performed with 3 biological replicates ± SEM. Statistical analysis was performed by the nonparametric multiple comparisons Steel test with a control group; *P < .05, **P < .01.