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Internalization of the thrombopoietin receptor is regulated by 2 cytoplasmic motifs
Debra D Dahlen1, Virginia C Broudy, Jonathan G Drachman
1Division of Hematology, University of Washington, Seattle, WA 98104, USA. debrad@u.washington.edu
Abstract:
Receptor-mediated internalization appears to be the primary mean of regulating the plasma level of thrombopoietin (TPO). However, the processes that regulate Mpl internalization have not previously been described. Using the cytokine-dependent cell line BaF3, we have identified 2 distinct motifs within the cytoplasmic domain of Mpl that underlie ligand-dependent internalization. Removal of the fourth cytoplasmic tyrosine residue by deletion or truncation results in a significant decrease in maximal internalization. The remaining receptor internalization is abrogated by deletion of cytoplasmic residues 54-69, which include the core box2 region (L54L55E56I57L58) and the only dileucine motifs (L54L55 and I57L58) within the cytoplasmic domain of Mpl. Receptor internalization mediated by this latter subdomain does not require Jak2 activation. Furthermore, TPO-stimulated cellular proliferation appears to be directly correlated with receptor internalization, indicating that internalization of the TPO/Mpl complex may be essential for normal signal transduction. Finally, we have demonstrated that upon removal of TPO from the supernatant, Mpl promptly reappears on the cell surface, suggesting that a pool of intracellular Mpl can be rapidly recycled to the cell surface. These data help identify the receptor motifs involved in TPO-induced internalization of Mpl and suggest that Mpl translocation may be necessary for normal cellular proliferation.
Insights
Ligand-dependent internalization of thrombopoietin (TPO) receptor Mpl is regulated by specific cytoplasmic motifs. Mpl recycling and internalization are crucial for TPO signaling and cellular proliferation.
Area of Science:
- Cell Biology
- Molecular Biology
- Hematology
Background:
- Receptor-mediated endocytosis is key for regulating plasma thrombopoietin (TPO) levels.
- Mechanisms governing Mpl internalization, the TPO receptor, remain largely uncharacterized.
Purpose of the Study:
- To identify specific motifs within the Mpl cytoplasmic domain responsible for ligand-dependent internalization.
- To investigate the role of Mpl internalization in TPO signaling and cellular proliferation.
Main Methods:
- Utilized the BaF3 cell line, a cytokine-dependent model system.
- Employed deletion and truncation mutagenesis of the Mpl cytoplasmic domain.
- Assessed receptor internalization, Jak2 activation, and cellular proliferation in response to TPO.
Main Results:
- Identified two distinct motifs in the Mpl cytoplasmic domain critical for TPO-induced internalization.
- Deletion of the fourth cytoplasmic tyrosine significantly reduced internalization.
- Abrogation of internalization by deleting residues 54-69, including dileucine motifs, occurred independently of Jak2 activation.
- TPO-stimulated proliferation correlated directly with Mpl internalization.
- Demonstrated rapid recycling of intracellular Mpl to the cell surface upon TPO removal.
Conclusions:
- Specific Mpl cytoplasmic motifs, including dileucine sequences, mediate TPO-dependent internalization.
- Mpl internalization is essential for TPO-mediated signal transduction and cellular proliferation.
- A readily recyclable pool of intracellular Mpl exists, facilitating rapid surface reappearance.