Functional Studies of the Extracellular Domain of Mpl

该提案的广泛，长期目标是了解细胞因子受体的细胞外结构和它们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。对这些过程的进一步了解最终将有助于开发血液病和其他疾病的新治疗策略。该提案旨在研究血小板生成素受体Mpl在配体结合和控制受体激活方面的结构/功能关系。本项目的具体目的是：1）检验Mpl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活化所需的特异性区域的假设。2)评估Mpl截短突变体导致组成性细胞生长的机制。3)验证Mpl的激活截短突变体可以在原代鼠骨髓细胞中发挥作用的假设。该项目在几个方面与健康有关。细胞因子受体的配体结合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展有关。Mpl的高水平表达与白血病和骨髓增生异常的不良预后相关，并且Mpl的异常表达与人和小鼠的骨髓增生性疾病相关。Mpl的结构/功能关系的研究将提供深入了解这种受体如何有助于正常造血和血液系统疾病的发展。识别Mpl的特定区域，所需的配体结合和受体活化将有助于设计新的治疗剂，可以特异性抑制Mpl功能。研究设计和方法包括使用分子建模和PCR介导的诱变来定位Mpl胞外结构域内的功能区域。逆转录病毒转导培养的细胞系和小鼠骨髓细胞与突变体和全长形式的Mpl将被用来确定参与受体激活和下游效应的激活突变的机制。

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.