The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis

Abstract

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...

CRLF2 over-Expression Is a Poor Prognostic Marker in Children with High Risk T-Cell Acute Lymphoblastic Leukemia

“…Further biological studies should be afforded to exploit the CRLF2 pathway in T-ALL. Interestingly, it was recently reported in the literature that the activity of another cytokine receptor (cMPL) did not depend on its cell surface expression [31]. The authors assumed that the receptor with an abnormal subcellular distribution may be active and particularly sensitive to the low amount of ligand that may enter into the cell through trace levels of the receptor on the cell surface.…”

Genetic Alterations of the Thrombopoietin/MPL/JAK2 Axis Impacting Megakaryopoiesis

“…Sometimes, mild thrombocytosis was observed in heterozygous cases. Later on, it was reported that MPL P106L presents a defect in trafficking that prevents its translocation to the cell surface, but is still capable to bind TPO and to signal in the cytosol ( 62 ). This seems unlikely since the extracellular domain of MPL is localized in the lumen of endoplasmic reticulum.…”

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

“…13 It is important to determine the functional impact of CAMT mutations on Mpl signaling and trafficking, because clinical presentation, disease progression, and treatment options reflect the underlying cellular mechanisms. 6,14,15 In this study, the severity of CAMT type I disease in 3 siblings was associated with a homozygous double MPL K39N/W272R mutant that resulted in complete blocking of Mpl trafficking to the plasma membrane. Currently, HSC transplantation is the only curative option for pediatric patients with life-threatening CAMT.…”

“…Since the first description of a disease-associated MPL mutation in CAMT in 1999, 1 more than 50 different genetic events have been reported for MPL, 2,3 and they are sometimes associated with defects in surface presentation. [4][5][6] Notably, the MPL Baltimore substitution (K39N) is associated with high platelet counts in patients of African American descent, despite incomplete processing and reduced Mpl protein levels. 7 Although cell surface expression of Mpl is required for stimulation by its ligand (Tpo), complex relationships between mutant and wildtype (WT) forms of Mpl, Jak2, and the endoplasmic reticulum (ER) chaperone calreticulin govern both the intracellular trafficking of receptors and signal propagation.…”