Functional Studies of the Extracellular Domain of Mpl

这项建议的广泛和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。增加对这些过程的了解最终将有助于开发针对血液病和其他疾病的新治疗策略。这项建议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。本项目的具体目的是：1)验证MPL胞外区的膜-远端细胞因子受体同源模块(CRM)包含配体结合所需的特定区域并防止受体自发激活的假设。2)评价MPL截短突变体导致构成细胞生长的机制。3)验证激活MPL截断突变体可以在原代小鼠骨髓细胞中发挥作用的假设。这个项目在几个方面与健康有关。细胞因子受体与配体结合的缺陷可能导致疾病的发展。结构性激活的受体与白血病和骨髓增生性疾病的发生有关。在白血病和骨髓异常增生症中，MPL的高水平表达与预后不良有关，在人类和小鼠中，MPL的异常表达与骨髓增生性疾病有关。对MPL结构/功能关系的研究将有助于深入了解该受体如何促进正常的造血和血液病的发展。识别配体结合和受体激活所需的MPL特定区域将有助于设计能够特异性抑制MPL功能的新的治疗药物。研究设计和方法包括利用分子模拟和聚合酶链式反应介导的突变来定位MPL胞外区的功能区域。将利用逆转录病毒转导培养的细胞系和携带突变和全长MPL的小鼠骨髓细胞来确定受体激活的机制以及激活突变的下游影响。

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.