Figure 1.
Activation of TpoR signaling by L498W-H499C mutations. (A) Effects of H499C and L498W mutations on TpoR signaling. HEK-293T cells were transiently transfected with the indicated hemagglutinin (HA)-TpoR variants, along with complementary DNAs (cDNAs) coding for JAK2, STAT5, and SpiLuc Firefly luciferase reporter reflecting STAT5 transcriptional activity and normalized with a control reporter (pRL-TK) containing Renilla luciferase. Shown are averages of 3 independent experiments, each performed in triplicate + standard deviation (SD; error bars). (B) Induction of autonomous proliferation of Ba/F3 cells by L498W and L498W-H499C mutants of TpoR. Ba/F3 cells stably expressing the TpoR mutants of interest were grown in the absence of cytokine. Cell proliferation was assessed by CellTiter-Glo (CTG) luminescent cell viability assay. (C) H499C increased cell-surface localization of TpoR. HEK-293T cells were transiently transfected with the indicated HA-TpoR variants. TpoR cell-surface localization among fluorescein isothiocyanate (FITC)–positive cells was assessed by flow cytometry using anti-TpoR antibody. Shown is 1 representative of 2 experiments. (D) TpoR dimerization in the absence of cytokine. Dimerization of TpoR was determined in HEK-293T using the NanoBiT luciferase protein fragment complementation assay using Nano-luciferase fragments fused in frame with the cytosolic domain of TpoR. (E) The extracellular domain of TpoR was not needed for constitutive activation by the L498W-H499C mutant. HEK-293T cells were transiently transfected with the indicated HA-TM-intracellular (TM-IC) TpoR variants, along with cDNAs coding for JAK2, STAT5, and SpiLuc Firefly luciferase reporter reflecting STAT5 transcriptional activity and normalized with a control reporter (pRL-TK) containing Renilla luciferase. Shown are averages of 4 independent experiments, each performed in triplicate + SD (error bars). (F) TpoR H499C continued to be overexpressed after cycloheximide (CHX) treatment. Ba/F3 cells stably expressing the TpoR mutants of interest were grown in presence of interleukin-3 and treated with CHX (50 μg/mL). TpoR cell-surface localization among FITC+ cells was assessed by flow cytometry using anti-TpoR antibody. Upper panel is an average of 3 independent experiments + SD (error bars). Lower panel is 1 representative of 3 experiments. (G) Model of the monomeric WT and dimeric L498W TM domains (TMDs) of the TpoR. On the left, the monomeric TMD has a break in the helical secondary structure in the region N-terminal to H499.12 On the left, in the L498W-active TMD dimer, the helix is induced in the region N-terminal to H499. *P < .05, **P < .01, ***P < .001. EC, extracellular; Elt, eltrombopag; eV, empty vector; LgBit, Nano-luciferase large-bit subunit; ns, nonsignificant (nonparametric multiple-comparison Steel test with control); PE, phycoerythrin; PIG, pMX-IRES-GFP vector; SmBit, Nano-luciferase small-bit subunit.

Activation of TpoR signaling by L498W-H499C mutations. (A) Effects of H499C and L498W mutations on TpoR signaling. HEK-293T cells were transiently transfected with the indicated hemagglutinin (HA)-TpoR variants, along with complementary DNAs (cDNAs) coding for JAK2, STAT5, and SpiLuc Firefly luciferase reporter reflecting STAT5 transcriptional activity and normalized with a control reporter (pRL-TK) containing Renilla luciferase. Shown are averages of 3 independent experiments, each performed in triplicate + standard deviation (SD; error bars). (B) Induction of autonomous proliferation of Ba/F3 cells by L498W and L498W-H499C mutants of TpoR. Ba/F3 cells stably expressing the TpoR mutants of interest were grown in the absence of cytokine. Cell proliferation was assessed by CellTiter-Glo (CTG) luminescent cell viability assay. (C) H499C increased cell-surface localization of TpoR. HEK-293T cells were transiently transfected with the indicated HA-TpoR variants. TpoR cell-surface localization among fluorescein isothiocyanate (FITC)–positive cells was assessed by flow cytometry using anti-TpoR antibody. Shown is 1 representative of 2 experiments. (D) TpoR dimerization in the absence of cytokine. Dimerization of TpoR was determined in HEK-293T using the NanoBiT luciferase protein fragment complementation assay using Nano-luciferase fragments fused in frame with the cytosolic domain of TpoR. (E) The extracellular domain of TpoR was not needed for constitutive activation by the L498W-H499C mutant. HEK-293T cells were transiently transfected with the indicated HA-TM-intracellular (TM-IC) TpoR variants, along with cDNAs coding for JAK2, STAT5, and SpiLuc Firefly luciferase reporter reflecting STAT5 transcriptional activity and normalized with a control reporter (pRL-TK) containing Renilla luciferase. Shown are averages of 4 independent experiments, each performed in triplicate + SD (error bars). (F) TpoR H499C continued to be overexpressed after cycloheximide (CHX) treatment. Ba/F3 cells stably expressing the TpoR mutants of interest were grown in presence of interleukin-3 and treated with CHX (50 μg/mL). TpoR cell-surface localization among FITC+ cells was assessed by flow cytometry using anti-TpoR antibody. Upper panel is an average of 3 independent experiments + SD (error bars). Lower panel is 1 representative of 3 experiments. (G) Model of the monomeric WT and dimeric L498W TM domains (TMDs) of the TpoR. On the left, the monomeric TMD has a break in the helical secondary structure in the region N-terminal to H499.12  On the left, in the L498W-active TMD dimer, the helix is induced in the region N-terminal to H499. *P < .05, **P < .01, ***P < .001. EC, extracellular; Elt, eltrombopag; eV, empty vector; LgBit, Nano-luciferase large-bit subunit; ns, nonsignificant (nonparametric multiple-comparison Steel test with control); PE, phycoerythrin; PIG, pMX-IRES-GFP vector; SmBit, Nano-luciferase small-bit subunit.

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