Characterizing phosphorylation-dependent regulation of metabotropic glutamate receptors by middle-down mass spectrometry

通过中下质谱表征代谢型谷氨酸受体的磷酸化依赖性调节

• 批准号: 10569501
• 负责人: Ashley Nichole Ives
• 金额: $ 4.31万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10569501
• 关键词: Arrestins; Bar Codes; Biochemical; Cells; Central Nervous System; Collaborations; Complex; Confocal Microscopy; Data; Data Analyses; Disease; Epilepsy; Eukaryota; Family; Fragile X Syndrome; Future; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutamate Receptor; Goals; Grant; Heterogeneity; Heterotrimeric GTP-Binding Proteins; Human; Ligands; Link; Literature; Location; Maps; Mass Spectrum Analysis; Mediating; Membrane; Metabotropic Glutamate Receptors; Methodology; Methods; Microscopy; Modification; Monitor; Mood Disorders; Mutagenesis; Nature; Neurons; Outcome; Pattern; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological Processes; Positioning Attribute; Proteins; Proteomics; Psychoses; Radiolabeled; Regulation; Research; Research Personnel; Series; Serine; Signal Transduction; Signaling Protein; Site; Site-Directed Mutagenesis; Synapses; Synaptic Transmission; Synaptic plasticity; System; Techniques; Testing; Threonine; Time; Training; Tyrosine; Visualization; Work; Writing; addiction; blind; career; combinatorial; desensitization; innovation; instrumentation; live cell imaging; metabotropic glutamate receptor 2; mutant; nervous system disorder; novel; protein purification; receptor; receptor function; receptor internalization; response; scaffold; skills; stoichiometry; therapeutic target; trafficking

ABSTRACT G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in eukaryotes and mediate a wide range of physiological processes. Extensive evidence has shown that GPCRs have complex signaling repertoires, and signal through multiple effector molecules beyond the canonical heterotrimeric G protein. It is believed that constitutive and ligand-induced GPCR phosphorylation is a key regulator in their complex signaling and modulation, and aberrations in receptor phosphorylation have been linked to the manifestation and persistence of disease states. Biochemical evidence suggests that these phosphorylation states are likely combinatorial in nature and highly heterogenous; however, mapping GPCR phosphorylation states remains out of reach given the limitations of common proteomics techniques which are low-throughput or blind to combinatorial information. Herein, we propose an innovative strategy to use intact protein mass spectrometry to characterize and quantify phosphorylation states of metabotropic glutamate receptor 2 which will allow for direct visualization of stoichiometric and positional phosphorylation heterogeneity that has previously been inaccessible. The central hypothesis of this proposal is that mGluR2 is subject to constitutive and ligand-induced phosphorylation that is highly heterogenous and these phosphorylations mediate several key aspects of mGluR2 function including mGluR2-mediated G protein signaling and receptor internalization. To test this hypothesis, we will use a combination of intact protein mass spectrometry and bottom-up proteomics to characterize phosphorylation states of mGluR2 in response to various ligands in a heterologous expression system (Aim 1). Based on the phosphorylation states discovered in Aim 1, we will then perform site-directed mutagenesis of mGluR2 to determine the impact of these phosphorylation states upon downstream G protein signaling and receptor internalization (Aim 2). To our knowledge, this study will be the first available demonstration of intact protein mass spectrometry to determine the identity and function of intact GPCR phosphorylation states; this will have broader implications as the approaches are generalizable to any receptor. The proposed work will be completed in collaboration between the Kelleher and Vafabakhsh groups and provide comprehensive training in cutting edge mass spectrometry-based proteomics, confocal microscopy, and traditional biochemical techniques. Both labs are respective experts in the fields of mass spectrometry and microscopy of GPCRs and are equipped with the instrumentation and expertise to support my training plan. I have constructed the training plan in close collaboration with Drs. Kelleher and Vafabakhsh to develop skills in targeted proteomics, advanced data analysis, live cell imaging via confocal microscopy, traditional biochemical techniques, scientific writing, and scientific presentation. These skills will afford me the opportunity to become an independent researcher and conduct hypothesis driven explorations of proteins and their modification states in my future career.

摘要 G蛋白偶联受体(GPCRs)是真核生物中最大的膜受体家族。 广泛的生理过程。大量证据表明，GPCR具有复杂的信号转导 并通过多个效应分子传递信号，而不是标准的异源三聚体G蛋白。它是 认为结构性和配基诱导的GPCR磷酸化是其复杂信号转导的关键调节因子 而受体磷酸化中的调节和异常与表现和 疾病状态的持续性。生化证据表明，这些磷酸化状态很可能 本质上是组合的，高度异质性；然而，绘制gpr磷酸化状态的图谱仍然存在。 考虑到普通蛋白质组学技术的局限性，这些技术是低通量的或盲目的 组合信息。在这里，我们提出了一种创新的策略，使用完整的蛋白质质谱学来 表征和量化代谢性谷氨酸受体2的磷酸化状态，这将允许直接 化学计量比和位置磷酸化异质性的可视化 无法接近。这一提议的中心假设是mGluR2受结构性和配基诱导的影响 高度异质性的磷酸化，这些磷酸化介导了mGluR2的几个关键方面 功能包括mGluR2介导的G蛋白信号转导和受体内化。为了检验这一假设，我们 将使用完整的蛋白质质谱学和自下而上的蛋白质组学的组合来表征 异源表达系统中mGluR2对不同配体的磷酸化状态(目标1)。 基于在目标1中发现的磷酸化状态，然后我们将执行定点突变 MGluR2来确定这些磷酸化状态对下游G蛋白信号的影响，以及 受体内化(目标2)。据我们所知，这项研究将是第一个完整的 蛋白质质谱仪，以确定完整的gpr磷酸化状态的特性和功能；这将 具有更广泛的含义，因为这些方法可以推广到任何受体。 拟议的工作将在Kelleher和Vafabakhsh小组之间的协作下完成，并提供 全面培训基于尖端质谱学的蛋白质组学、共聚焦显微镜和 传统的生化技术。两个实验室都是质谱学领域的专家， 他们配备了支持我的培训计划的仪器和专业知识。我 与Kelleher博士和Vafabakhsh博士密切合作制定了培训计划，以提高 靶向蛋白质组学、高级数据分析、共聚焦显微镜活细胞成像、传统生化 技术、科学写作和科学陈述。这些技能将使我有机会成为 独立研究人员并进行假设驱动的蛋白质及其修饰状态的探索 在我未来的职业生涯中。