Functional Studies of the Extracellular Domain of Mpl

本研究的长远目标是了解细胞因子受体的细胞外结构与其在正常造血中的功能之间的关系，以及这种结构的变化如何导致疾病的发展。增加对这些过程的了解将最终有助于开发新的血液疾病和其他疾病的治疗策略。本课题旨在研究血小板生成素受体Mpl在配体结合和受体激活控制中的结构/功能关系。该项目的具体目的是：1)验证Mpl细胞外结构域的膜-远端细胞因子受体同源模块（CRM）包含配体结合和防止自发受体激活所需的特定区域的假设。2)评估Mpl截断突变体导致本构细胞生长的机制。3)验证激活Mpl截断突变体在小鼠原代骨髓细胞中起作用的假设。这个项目在几个方面与健康有关。细胞因子受体结合配体缺陷可导致疾病发展。组成性激活受体与白血病和骨髓增生性疾病的发生有关。Mpl的高水平表达与白血病和骨髓增生不良预后相关，而Mpl的异常表达与人类和小鼠的骨髓增生性疾病相关。研究Mpl的结构/功能关系将有助于深入了解该受体如何促进正常造血和血液系统疾病的发展。确定Mpl中配体结合和受体激活所需的特定区域将有助于设计能够特异性抑制Mpl功能的新治疗剂。研究设计和方法包括使用分子模型和pcr介导的诱变来绘制Mpl细胞外结构域内的功能区。携带突变型和全长型Mpl的培养细胞系和小鼠骨髓细胞的逆转录病毒转导将被用来确定受体激活的机制和激活突变的下游效应。

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.