Novel Regulators of GPCR Signaling in Hematopoietic Stem Cells

项目摘要 造血干细胞和祖细胞（HSPC）是自我更新的可移植细胞，可支持终身免疫。 血液生产。HSPC功能和移植与通过G蛋白偶联的信号转导有关 受体（GPCR），特别是调节归巢和生态位保持的CXCR 4的受体。我们的理解 这些细胞中缺乏基本的GPCR调节机制。我们的实验室最近强调了这一点 发现两种GPCR运输蛋白GPRASP 1和GPRASP 2作为HSPC的负调节剂， 移植我们的初步数据进一步表明，这些蛋白质限制HSPC的静止和抗性 以CXCR 4依赖的方式诱导细胞凋亡。GPRASP 1和GPRASP 2在神经系统中被描述为 促进溶酶体降解和阿片类药物、多巴胺和其他选择的功能下调 受体。HSC中GPRASP 1和GPRASP 2调节的受体目前尚不清楚。担任博士后研究员 在McKinney-Freeman实验室，我将研究GPRASP 1和GPRASP 2在HSPCs中的分子功能。 在目的1中，我将使用生物化学方法来确定GPRASP 1和GPRASP 2对CXCR 4信号传导的影响。的 GPRASP 1和GPRASP 2对CXCR 4内吞、降解和下游信号转导结果的影响 将在小鼠HSPC中分析激动剂刺激。在目标2中，我将探讨GPRASP 1和GPRASP 2在 移植从脐带血分离的人HSPC。通过以下方法进行基因编辑的人类HSPC CRISPR/Cas9核转染以敲除GPRASP 1或GPRASP 2，并将用于竞争性异种移植 测定。通过跟踪野生型和敲除人HSPC的相对植入和血液产生， 异种移植后，将确定GPRASP蛋白对该过程的影响。在目标3中，我将 使用无偏倚的蛋白质组学方法研究GPRASP 1和GPRASP 2的受体靶点，以及GPRASP 1和GPRASP 2的受体靶点。 与这些蛋白质相关的下游运输途径。生物素邻近标记将使用 GPRASP-APEX融合蛋白在K562慢性粒细胞白血病细胞中的表达 激动剂刺激后的时间点。生物素化样品将进行串联质量标记和多重标记 质谱法以确定跨时间点的蛋白质的相对丰度。这将允许 与GPRASP 1相关的受体靶点和下游运输途径的生物信息学分析 和-2。将通过小鼠HSPC中的免疫荧光验证所有感兴趣的蛋白质缔合。这些 目标是利用我以前作为生物化学家的培训，但在HSPC领域提供丰富的培训机会 生物学麦金尼-弗里曼实验室和圣裘德是接受培训的理想环境， HSPC生物学。提供机构资源，包括最先进的流式细胞术和蛋白质组学核心， 将大大有助于这个项目的成功。此外，我将利用众多的职业 在我的奖学金期间发展资源。这些资源包括科学写作研讨会，演示文稿 和网络机会，以及教学和指导经验。

Project Summary Hematopoietic stem and progenitor cells (HSPCs) are self-renewing, transplantable cells that support lifelong blood production. HSPC function and transplantation are tied to signal transduction through G-protein coupled receptors (GPCRs), particularly that of CXCR4 which regulates homing and niche retention. Our understanding of basic GPCR regulatory mechanisms in these cells are lacking. This is highlighted by our laboratories recent finding that two GPCR-trafficking proteins, GPRASP1 and GPRASP2, act as negative regulators of HSPC transplantation. Our preliminary data further indicate that these proteins limit HSPC quiescence and resistance to apoptosis in a CXCR4-dependent manner. GPRASP1 and -2 have been described in the nervous system to promote the lysosomal degradation and functional downregulation of opioid, dopamine, and other select receptors. The receptors regulated by GPRASP1 and -2 in HSCs are currently unknown. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the molecular functions of GPRASP1 and -2 in HSPCs. In Aim 1, I will use biochemical methods to definethe impact of GPRASP1 and -2 on CXCR4 signaling. The impact of GPRASP1 and -2 on CXCR4 endocytosis, degradation, and downstream signaling outcomes after agonist stimulation will be analyzed in mouse HSPCs. In Aim 2, I will probe the role of GPRASP1 and -2 in transplantation of human HSPCs isolated from cord blood. Human HSPCs that have undergone gene editing by CRISPR/Cas9 nucleofection to knockout GPRASP1 or -2 and will be used in competitive xenotransplantation assays. By following the relative engraftment and blood production of wild type and knockout human HSPCs after xenotransplantation the impact of GPRASP proteins on this procedure will be determined. In Aim 3, I will use an unbiased proteomics approach to investigate the receptor targets of GPRASP1 and -2, as well as the downstream trafficking pathways associated with these proteins. Biotin proximity labeling will be performed using GPRASP-APEX fusion proteins in K562 chronic myelogenous leukemia cells both before and at multiple timepoints following agonist stimulation. Biotinylated samples will undergo Tandem Mass Tagging and multiplex mass spectrometry to determine the relative abundance of proteins across timepoints. This will allow for bioinformatic analysis of the receptor targets and downstream trafficking pathways associated with GPRASP1 and -2. All protein associations of interest will be validated by immunofluorescence in mouse HSPCs. These aims draw on my prior training as a biochemist but provide abundant training opportunities in the field of HSPC biology. The McKinney-Freeman laboratory and St. Jude are ideal environments in which to receive training in HSPC biology. Institutional resources provided, including state-of-the-art flow cytometry and proteomics cores, will greatly contribute to the success of this project. In addition, I will take advantage of numerous career development resources during my fellowship. These resources include scientific writing workshops, presentation and networking opportunities, and teaching and mentoring experiences.