Key Points• The c-Mpl activity in downstream signaling and in platelet homeostasis can be functionally separated.• The c-Mpl platelet homeostasis depends on correct processing and surface expression of the receptor, whereas downstream signaling does not.The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis. (Blood. 2015;125(7):1159-1169) IntroductionPlatelet production is stimulated by the interaction of the cytokine thrombopoietin (THPO) with its receptor c-Mpl on megakaryocytes and their progenitors with subsequent activation of several downstream pathways, including the Janus kinase/signal transducer and activator of transcription pathway, the phosphatidylinositol 3-kinase pathway, and the mitogen-activated protein kinase pathway. The homeostasis of platelet numbers in the blood is maintained by a negative feedback loop, which requires the clearance of THPO from the plasma by c-Mpl-carrying megakaryocytes and platelets.1 In addition, disruption of the signaling pathway by inactivating THPO or c-Mpl mutations can cause serious thrombocytopenia.2-4 By contrast, activating THPO or c-Mpl mutations can cause hereditary thrombocythemia (HT). [5][6][7][8][9][10][11][12] Interestingly, decreased expression of c-Mpl can also result in HT by high THPO levels, although the mechanism for this observation remains unknown. [13][14][15] Genotype analyses in HT have previously identified the transmembrane S505N, the extracellular N35K, and the P106L c-Mpl mutations to occur in Japanese, African American and Arabic populations, respectively. 5,9,12,[16][17][18] Furthermore, the c-Mpl S505N mutation in the transmembrane region and the juxtamembrane c-Mpl W515L and W515...
Thrombopoiesis is tightly regulated by the binding of thrombopoietin (THPO) to its receptor c-Mpl that leads to the clearance of THPO from the plasma thus establishing a negative feedback loop. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We focused on a comparison of gain-of-function mutation in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur, with the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations are known to also promote myeloproliferative malignancies and AML, whereas the P106L mutation causes hereditary thrombocytosis without a known predisposition to hematologic malignancies. We have now performed functional analyses of these gain-of-function mutations to address the question of how the different propensity to induce malignancy can be explained. We first analyzed the post-translational processing of the normal and the P106L mutated receptor in comparison to the receptors carrying the S505N, W515K and W515L mutations in transfected HeLa and BA/F3 cells. The normal and the S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The plasma membrane expression, assayed by confocal microscopy and FACS, of the S515N, W515K and W515L mutated receptors was comparable to the normal receptor, whereas the P106L mutated receptor was not detectable on the cell surface. Functional analyses of the THPO/c-Mpl signaling pathways in THPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the normal receptor gene and the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways. These results show that (1) the activation of c-Mpl by THPO does not absolutely require surface expression of the receptor (2) different c-Mpl gain-of-function mutations activate separable downstream pathways and (3) the predominant activation of the PI3K/m-TOR and Stat3/5 pathways correlates with the propensity to induce hematopoietic malignancy. Further, it is interesting to note that the c-Mpl P106L gain-of-function mutant is known to cause 10 to 20-fold elevated thrombopoietin (THPO) plasma levels in patients and is shown here not to be properly glycosylated and transported to the cell surface. By contrast, patients with the other gain-of-function mutations analyzed here show normal or even reduced THPO plasma concentrations and normal post-translational processing and cell surface expression. In conclusion, we propose that c-Mpl gain-of-function mutations exert their effect by predominantly activating either the PI3K/m-TOR and Stat3/5 or the ERK signaling pathways and that the predominant activation of PI3K/m-TOR and Stat3/5 correlates with the propensity to induce malignancy. Furthermore, THPO clearance and maintenance of the negative feedback loop regulating THPO plasma levels, but not signaling activity of the c-Mpl receptor necessarily require proper glycosylation, intracellular trafficking and cell surface expression. Disclosures No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.