Active and inactive conformations of human TpoR. (A) Illustration of the Put3-hTpoR TM-ICD fusion protein allowing controlled dimerization of hTpoR TM interfaces. The illustration represents the Put3 dimerization motif (wheat) fused to hTpoR TM (cyan) and ICD (magenta). Positions a and b are colored in yellow and red, respectively. The structure was generated using AlphaFold 2.0.³² The helical wheel diagram represents the amino acid in each of the seven possible positions, denoted as a to g. The amino acids in a and d are at the interface. TM, transmembrane; ICD, intracellular domain. (B) Helical wheel diagram of cc-hTpoR constructs with amino acids in the interface for each of the 7 cc-hTpoR. The active (green) and inactive (red) interfaces are highlighted. (C) Proliferation assay of Ba/F3 cells stably transduced with the indicated constructs. Values represent the mean (±SD) of 3 independent experiments performed in triplicate (N = 3, n = 9). (D) Representative flow cytometry measurement of STAT5 phosphorylation in Ba/F3 cells expressing the indicated constructs. Mean fluorescence intensity (MFI) is indicated. (E) Luminescent viability assay (CellTiter-Glo) used to measure proliferation of TF-1 cells stably transduced with indicated constructs. Values represent the mean (±SD) of 8 biological replicates (n = 8). (F) Representative flow cytometry measurement of STAT5 phosphorylation in TF-1 cells expressing the indicated constructs. Mean fluorescence intensity (MFI) is indicated. (G) STAT5 transcriptional activity was measured by a luciferase assay in HEK293T transiently transfected with hJAK2 WT and the indicated constructs. Values represent the mean (±SD) of 4 independent experiments performed in triplicate. (C,E,G) Statistics: two-way analysis of variance followed by the SIDAK multiple comparison test. ns, nonsignificant. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.