Running title: c-Mpl P106L receptor processing and activation Category: Platelets and thrombopoiesis The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis

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 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 thrombocythemia 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 following THPO treatment in Ba/F3 cells. Despite their opposite function, both, R102P and P106L lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation and to elevated THPO serum levels in affected patients. These findings indicate that the activation of downstream signaling by cMpl P106L does not require correct processing, trafficking and cell surface expression of cMpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. We thus propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis. For personal use only. on June 4, 2017. by guest www.bloodjournal.org From