Role of the Distal Half of the c-Mpl Intracellular Domain in Control of Platelet Production by Thrombopoietin In Vivo

Abstract

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Δ60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Δ60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

ACKNOWLEDGMENTS

We thank the Genentech DNA synthesis and DNA sequencing and ES cell microinjection laboratories; J. Flores for help with animal handling; D. Eaton, P. Fielder, and S. Bunting for advice and support; N. Ghilardi and R. Skoda for comments on the manuscript; R. Ashman and S. Lucas of the St Jude Flow Cytometry Facility for their advice and expertise; and S. Steward of the St Jude Division of Experimental Hematology for preparation of bone marrow cell suspensions.