Functional Studies of the Extracellular Domain of Mpl

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The broad, long-term objectives of this proposal are to understand the relationship between the extracellular structure of cytokine receptors and their function in normal hematopoiesis and how changes in this structure can lead to the development of disease. Increased understanding of these processes will ultimately contribute to the development of new therapeutic strategies for hematologic disorders and other diseases. This proposal is designed to study the structure/function relationships of Mpl, the thrombopoietin receptor, in ligand binding and in control of receptor activation. The specific aims of this project are: 1) Test the hypothesis that the membrane-distal cytokine receptor homology module (CRM) of the extracellular domain of Mpl contains specific regions that are required for ligand binding and to prevent spontaneous receptor activation. 2) Evaluate the mechanisms through which truncation mutants of Mpl lead to constitutive cell growth. 3) Test the hypothesis that activating truncation mutants of Mpl can function in primary murine marrow cells. This project is health-related in several ways. Defective ligand binding by cytokine receptors can lead to disease development. Constitutively activated receptors are associated with development of leukemias and myeloproliferative disorders. High levels of expression of Mpl are associated with a poor prognosis in leukemia and myelodysplasia, and abnormal expression of Mpl is associated with myeloproliferative disease in humans and in mice. Study of the structure/function relationships of Mpl will provide insights into how this receptor contributes to normal hematopoiesis and to the development of hematologic disorders. Identification of specific regions of Mpl that are required for ligand binding and receptor activation will contribute to the design of new therapeutic agents that can specifically inhibit Mpl function. The research design and methods include use of molecular modeling and PCR-mediated mutagenesis to map the functional regions within the extracellular domain of Mpl. Retroviral transduction of cultured cell lines and murine marrow cells with mutant and full-length forms of Mpl will be utilized to determine the mechanisms involved in receptor activation and the downstream effects of activating mutations.