G-CSF Receptor and Ubiquitination

描述（由申请人提供）：粒细胞生成的一个显著特征是调节中性粒细胞（PMN）的产生和释放，以维持循环中的稳态水平。粒细胞集落刺激因子受体（G-CSFR）作为髓系细胞增殖和分化的主要调节剂以及作为PMN和干细胞动员的调节剂在此过程中起关键作用。G-CSFR表达和信号传导的失调与中性粒细胞缺乏和人类白血病有关。尽管G-CSFR在调节粒细胞生成中起关键作用，并且目前广泛临床使用G-CSF来预防和/或治疗中性粒细胞减少症和用于干细胞动员，但下调G-CSFR表达和信号传导以防止不受控制的骨髓细胞扩增的分子机制仍然未知。这项建议的总体目标是确定泛素化在粒细胞集落刺激因子受体（G-CSFR）失活中的作用。我们推测，G-CSFR的泛素化修饰调节其在细胞表面的表达及其在细胞内的命运和信号转导能力，并且G-CSFR泛素化的缺陷有助于人类疾病。我们将在本申请中提出的研究中使用超微结构分析以及分子，生物化学和功能方法来分析G-CSFR泛素化对受体运输和信号传导的动力学来验证我们的假设。我们还将调查是否强制泛素化的内化能力不强的突变体G-CSFR形式从急性髓细胞性白血病（AML）患者纠正异常受体表面表达和贩运，以扭转过度增殖表型。将G-CSFR变体逆转录病毒转导到来自G-CSFR缺失（-/-）小鼠的原代细胞中将允许我们确认在细胞系中获得的结果并建立其生理相关性。确定G-CSFR泛素化的功能后果将为G-CSFR脱敏的基本机制提供新的见解，其可能具有调节粒细胞生成和祖细胞动员的治疗应用。

DESCRIPTION (provided by applicant): A remarkable feature of granulopoiesis is the regulated production and release of neutrophils (PMN) to maintain homeostatic levels in the circulation. The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in this process as the major regulator of myeloid cell proliferation and differentiation and as a modulator of PMN and stem cell mobilization. Deregulation in G-CSFR expression and signaling is linked to neutrophil deficiencies and human leukemias. Despite the critical role that the G-CSFR plays in regulating granulopoiesis and the current widespread clinical use of G-CSF for prevention and/or treatment of neutropenia and for stem cell mobilization, the molecular mechanisms that downregulate G-CSFR expression and signaling to prevent uncontrolled myeloid cell expansion remain unknown. The overall goal of this proposal is to determine the role of ubiquitination in inactivation of the granulocyte colony-stimulating factor receptor (G-CSFR). We hypothesize that ubiquitin-modification of the G-CSFR regulates its expression at the cell surface and its intracellular fate and signaling potency and that defects in G-CSFR ubiquitination contribute to human disease. We will test our hypothesis in the studies proposed in this application using ultrastructural analyses as well as molecular, biochemical, and functional approaches to analyze the dynamics of G-CSFR ubiquitination on receptor trafficking and signaling. We will also investigate whether forced ubiquitination of internalization-incompetent mutant G-CSFR forms from patients with acute myelogenous leukemia (AML) corrects aberrant receptor surface expression and trafficking to reverse the hyperproliferative phenotype. Retroviral transduction of G-CSFR variants into primary cells from G-CSFR null (-/-) mice will allow us to confirm results obtained in cell lines and establish their physiological relevance. Determination of the functional consequences of G-CSFR ubiquitination will provide new insights into the fundamental mechanisms of G-CSFR desensitization which may have therapeutic application for modulation of granulopoiesis and progenitor cell mobilization.