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: 10388500
• 负责人: Ashley Nichole Ives
• 金额: $ 4.25万
• 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/10388500
• 关键词: Arrestins; Bar Codes; Biochemical; Cells; 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 Protein alpha Subunits, Gs; 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; 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; base; 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（GPCR）是真核生物中最大的膜受体家族，并介导细胞内的G蛋白偶联。 广泛的生理过程。大量证据表明，GPCR具有复杂的信号传导， 库，并通过多个效应分子的信号超出了典型的异源三聚体G蛋白。是 认为组成型和配体诱导的GPCR磷酸化是其复杂信号传导的关键调节因子 受体磷酸化中的调节和畸变与这种表现有关， 疾病状态的持续。生化证据表明，这些磷酸化状态可能是 组合的性质和高度异质性;然而，绘制GPCR磷酸化状态仍然是一个问题。 鉴于普通蛋白质组学技术的局限性，这些技术是低通量或盲目的， 组合信息在此，我们提出了一个创新的策略，使用完整的蛋白质质谱， 表征和量化代谢型谷氨酸受体2的磷酸化状态，这将允许直接 化学计量和位置磷酸化异质性的可视化， 难以接近这一提议的中心假设是mGluR 2受组成性和配体诱导的调节。 磷酸化是高度异质性的，这些磷酸化介导mGluR 2的几个关键方面 功能包括mGluR 2介导的G蛋白信号传导和受体内化。为了验证这个假设，我们 将使用完整蛋白质质谱和自下而上的蛋白质组学相结合， 在异源表达系统中响应于各种配体的mGluR 2的磷酸化状态（Aim 1）。 基于目标1中发现的磷酸化状态，我们将进行定点诱变， mGluR 2，以确定这些磷酸化状态对下游G蛋白信号传导的影响， 受体内化（Aim 2）。据我们所知，这项研究将是第一个完整的证明， 蛋白质质谱法来确定完整GPCR磷酸化状态的身份和功能;这将 具有更广泛的影响，因为这些方法可推广到任何受体。 拟议的工作将在凯莱赫集团和瓦法巴赫什集团的合作下完成， 在尖端质谱为基础的蛋白质组学，共聚焦显微镜，和 传统的生化技术。两个实验室都是质谱领域的专家， 我的团队拥有GPCR的显微镜，并配备了仪器和专业知识来支持我的培训计划。我 我与Kelleher博士和Vafabakhsh博士密切合作，制定了培训计划， 靶向蛋白质组学，先进的数据分析，通过共聚焦显微镜进行活细胞成像，传统的生物化学 技术、科学写作和科学陈述。这些技能将使我有机会成为 一个独立的研究人员和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。