An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor

Abstract

Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (delta5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, delta5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. Delta5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the delta5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces.

Figure 1.

TpoR contains a cytoplasmic KWQFP motif that maintains the unliganded receptor inactive. (A) The TpoR JM domain contains 5 additional residues that show no homology to the EpoR (shown in red italics). To construct theΔ5TpoR, the KWQFP motif in the TpoR cytosolic JM domain was deleted by PCR mutagenesis. (B) Proliferation assays performed in Ba/F3 cells expressing the wtTpoR or Δ5TpoR at equal GFP levels in the absence of any cytokines or stimulated with 5 ng/mL Tpo as indicated. Cell numbers (averages of triplicates ± SD) were counted at day 12. (C) Total protein levels of the wtTpoR or Δ5TpoR in Ba/F3 cells infected to GFP levels at 40% to 50%, as revealed by Western blotting using anti-HA antibodies. (D) HA staining for cell-surface levels of the wtTpoR (red) or Δ5TpoR (blue) in Ba/F3 or hematopoietic progenitor cells infected to GFP levels at 40%. Green indicates Ba/F3 cells (negative control).

Figure 2.

Constitutive and ligand-induced activation of Δ5TpoR in primary myeloid progenitors. (A) Bone marrow cells were infected with retroviruses coding for wtTpoR or Δ5TpoR and sorted for equivalent GFP before plating. Cell-surface levels of wtTpoR (red) or Δ5TpoR (blue) were determined by FACS with anti-HA antibodies. Green indicates Ba/F3 cells (negative control). (B) Colony formation of hematopoietic progenitor cells expressing the wtTpoR or Δ5TpoR in the absence of Tpo (- Tpo). (C) Colony formation of hematopoietic progenitor cells expressing the wtTpoR or Δ5TpoR in the presence of 10 ng/mL Tpo (+ Tpo). Shown in panels B and C are averages of colony numbers (averages of triplicates ± SD) from 1 representative experiment. Data were similar among 3 independent experiments.

Figure 3.

Constitutive and ligand-activated signaling by Δ5TpoR. Tyrosine phosphorylation of Jak2 and Tyk2 (A) and STAT5 and STAT3 (B) in cells expressing the wtTpoR or the Δ5TpoR was determined in Ba/F3 cells expressing the wtTpoR or Δ5TpoR at equal GFP and protein levels, and after selection with Tpo (for wtTpoR cells) or without any cytokines (for Δ5TpoR). Cells were stimulated or not with 50 ng/mL Tpo for 7 minutes. (A) Cells were lysed and immunoprecipitated with antiphosphotyrosine (P-Y 4G10) antibodies and analyzed by Western blotting with anti-P-Jak2 or anti-P-Tyk2 antibodies. Western blotting using anti-Jak2 or anti-Tyk2 antibodies, respectively, revealed total protein levels of cell lysates used for immunoprecipitations. (B) Western blotting using anti-P-STAT5 or anti-P-STAT3 antibodies revealed levels of STAT5 or STAT3 phosphorylation. Blots were stripped and analyzed for equal protein levels using anti-STAT5 or anti-STAT3 antibodies.

Figure 4.

Constitutive and ligand-induced transcriptional activation by Δ5TpoR. Ba/F3 cells selected in Tpo or without cytokines were starved for 3 hours in RPMI/1mg/mL BSA and electroporated with pGL3bPpr2-luc, pLHRE-luc, or pSRE-luc in order to measure transcriptional activity of STAT3 (A), STAT5 (B), or MAP kinase (C), respectively. pRL-TK encoding the renilla luciferase was coelectroporated for normalization of luciferase values. Cells were stimulated with 50 ng/mL Tpo or mock-treated as indicated. Cell lysates were prepared 2 hours after stimulation and analyzed for luciferase activity. Results shown here reflect averages of triplicate values ± SD. Similar results were obtained performing 3 independent experiments.

Figure 5.

Identification of K507 and W508 as the residues responsible for the inhibitory function of the KWQFP motif on the unliganded TpoR. (A) Proliferation assays of the TpoR mutants containing alanine substitutions were performed with Ba/F3 cells expressing equal GFP levels. Cells were treated with 5 ng/mL Tpo where indicated or grown in the absence of any cytokines. Cell numbers were counted at day 9. Results shown here reflect averages of triplicates ± SD from 1 representative experiment. (B) Model of inhibition by the cytoplasmic KWQFP motif of the activity of TpoR. In the presence of Tpo, the receptor adopts a conformation where the KWQFP motif cannot bind to membranes or membrane proteins and interdict signaling. (C) Colony formation induced by Tpo-activated AWQFP and KAQFP point mutants of the TpoR. Colonies were assayed as in Figure 2C.

Figure 6.

Solid-state NMR spectra of TpoR TM-JM peptides. Only the carbonyl region of the ¹³C spectrum is shown. The spectra of lipid alone (dashed line) exhibit only the resonance of the lipid acyl chain carbonyl due to the natural abundance of ¹³C. The specific ¹³C labels at the backbone carbonyl of L501, L503, L505, and L506 are observed at approximately 175 ppm (solid line), characteristic of helical secondary structure extending from L501 to F510.