The membrane-distal part of extracellular domain of TpoR (D1D2) and its associated N-glycosylation sites control CALR mutant activity. (A) Schematic representation of TpoR with its extracellular subdomains and the N-glycosylation sites that have been mutated into Q. The more distal subdomains of extracellular domain of TpoR, namely D1D2, contain 2 N-glycosylated sites annotated N1 (N117) and N2 (N178). The proximal subdomains D3D4 contain two N-glycosylated sites annotated N3 (N298) and N4 (N358). STAT5 transcriptional activity was assessed by luciferase assay in γ2A cells transiently expressing STAT5, JAK2, CALR wild-type, or mutants together with the following TpoR mutants: TpoR D1D2 (B), TpoR D3D4 (C), TpoR N1234tQ (D), TpoR N123tQ (E), TpoR N124tQ (F), TpoR N134tQ (G), TpoR N234tQ (H), and TpoR N12tQ (I). Mutant TpoR missing D1D2, rather than D3D4, is unable to support CALR mutant activity (B). 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.