YRRL Motifs in the Cytoplasmic Domain of the Thrombopoietin Receptor Mpl Regulate Receptor Internalization and Degradation.

Thrombopoietin (TPO), acting through its receptor Mpl, promotes survival and proliferation of hematopoietic progenitor cells and also drives megakaryocyte differentiation. The pro-proliferation and survival signals activated by TPO must therefore be tightly regulated to prevent uncontrolled cell growth. Several mechanisms to down-regulate hematopoietic growth factor signaling have been identified, including increased expression of suppressors of cytokine signaling (SOCS) proteins, activation of protein phosphatases, and endocytosis and degradation of activated growth factor receptors. In this work we determined the mechanisms that control TPO-stimulated Mpl internalization and defined the processes leading to Mpl degradation using IL-3-dependent BaF3 cells engineered to express wild type (WT) and mutant forms of human Mpl. Stimulation of BaF-Mpl cells with TPO lead to rapid endocytosis of Mpl which was blocked by pre-treatment with the clathrin-mediated endocytosis inhibitor monodansylcadaverine, indicating that Mpl internalization is clathrin-mediated. Additionally, we found that inhibition of Janus kinase 2 (Jak2) and Src-family kinases greatly reduced Mpl internalization. Sites of clathrin assembly on the plasma membrane contain the adaptor protein complex AP2, which associates with transmembrane proteins enabling targeted endocytosis. Association of AP2 and transmembrane proteins relies on an interaction between AP2μ2 subunit and internalization motifs YXXΦ (where X=any amino acid and Φ=bulky hydrophobic) expressed by target proteins. Mpl contains two YXXΦ motifs, at cytoplasmic Y8 (Y₈RRL) and Y78 (Y₇₈RRL). BaF-Mpl cells transfected with siRNA targeted to AP2 displayed greatly reduced TPO-mediated internalization of Mpl, demonstrating that AP2-Mpl association is critical for Mpl endocytosis. Next, we introduced Y to F point mutations at Mpl cytoplasmic Y8 and Y78 individually, and in combination (Y8+78F). Internalization was greatly reduced in Mpl Y78F and Mpl Y8+78F after TPO stimulation, whereas Mpl Y8F internalization was only slightly attenuated. Furthermore, we found that Mpl Y78F and Y8+78F exhibited increased proliferation and increased strength and duration of activated Jak2 and AKT in response to TPO, compared to Mpl WT and Y8F. In addition to mediating endocytosis, cytoplasmic YXXΦ motifs located between 6 and 9 residues from the transmembrane domain have been shown to mediate lysosomal targeting of proteins following endocytosis. Consequently, we studied the role of Mpl Y₈RRL motif in Mpl lysosome targeting. BaF-Mpl WT and Y8F cells were pretreated with cyclohexamide and stimulated with TPO for up to 2 hours. Mpl Y8F displayed greatly reduced Mpl degradation in response to TPO compared to WT. Furthermore, Mpl Y8F recycled back to the plasma membrane following TPO starvation quicker than WT Mpl, suggesting that the Mpl Y8F remains in endosomes, rather than being targeted to lysosomes. Our data shows that Mpl cytoplasmic YRRL motifs are responsible for both TPO-mediated internalization via interactions with AP2 and lysosomal targeting following endocytosis. These findings significantly advance our understanding of normal Mpl function. Further study of internalization motifs, which are present in receptors for other hematopoietic growth factors including; erythropoietin, granulocyte colony stimulating factor, leukemia inhibitory factor and interleukin, may highlight the importance of these sequences in mediating hematopoiesis and potentially aid identification of novel targets in hematological disease.