Age and pathology-induced alteration of G protein-coupled receptor signaling

年龄和病理诱导的 G 蛋白偶联受体信号传导改变

基本信息

批准号：
8552365
负责人：
Stuart Maudsley
金额：
$ 24.39万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8552365
关键词：
Acetylcholine Acute Affect Affinity Age Aging Aging-Related Process Alzheimer&apos s Disease Amyloid beta-Protein Antibodies Area Bees Biological Assay Butyric Acids Cell Line Cell membrane Cell surface Cells Chemicals Clathrin Confocal Microscopy Dendritic Spines Disease Disease model Environment Epitopes Focal Adhesions G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors Gated Ion Channel Glucose Glutamates Hemagglutinin Human Huntington Disease Hydrogen Peroxide Immune Sera In Vitro Integral Membrane Protein Investigation Life Ligands Link Mediating Membrane Membrane Microdomains Modeling Multiprotein Complexes Muscarinic Acetylcholine Receptor Muscarinics Nature Neuraxis Neurodegenerative Disorders Neurons Neurotransmitters Oxidative Stress Parkinson Disease Pathology Pharmacology Phase Process Production Protein Isoforms Proteins Proteomics Receptor Activation Receptor Mediated Signal Transduction Recruitment Activity Research Scaffolding Protein Sepharose Serum Signal Transduction Signaling Protein Site Structure Synapses Synaptic Transmission System Techniques Technology Therapeutic Tissues Transcript Work animal tissue base beta-arrestin coated pit deprivation desensitization gamma secretase human RIPK1 protein mimetics neurotransmitter release normal aging novel overexpression programs receptor receptor function scaffold spinophilin stoichiometry stressor tandem mass spectrometry therapeutic target

项目摘要

Research Summary My proposed study involves investigation of age- and disease-dependent alteration of three distinct levels of receptor-mediated signal transduction control. These three levels of control are: receptor accessory protein modulation of signal transduction; scaffolding protein functionality in neuronal cells; receptor microdomain environment and its impact on receptor function. To these multiple ends myself and my research fellows have been undertaking the following research to illuminate these areas. Receptor Accessory protein modulation We have generated two sets of stably expressing human neuronal cell lines (SH-SY5Y) expressing an epitope-tagged (3x haemagglutinins) human m1 and m2 muscarinic acetylcholine G protein-coupled receptors (GPCRs). These receptors have been chosen as they represent one of the most important and tractable therapeutic targets for the treatment of Alzheimers disease (AD), e.g. the only currently prescribed AD therapeutic, AriceptTM, works by facilitating stimulation of these targets. We have chosen 24 clones of each of these cell lines and have been employing these for proteomic research. Our need to generate these clones resides in the purification techniques required to isolate large amounts of the receptor proteins so that their associated accessory proteins can be identified by tandem mass spectrometry. Using conventional antibody-based techniques using sera directed against functional regions of the receptor it is likely that subtle ultrastructural changes between receptor isoforms may be disregarded. Our hypothesis is that during aging and disease there are changes of the stoichiometry of the receptor with its accessory proteins and that this then affects the interaction of the receptor with its cognate ligand. Therefore our need to unambiguously purify transmembrane receptors was paramount. We have successfully purified high numbers of muscarinic receptors and demonstrated that large numbers of accessory proteins can associate with the GPCR. We intend now to compare these associated proteins to the groups of proteins that interact with the receptor in a disease state model, i.e. addition of exogenous amyloid beta peptide or oxidative stress in the form of either hydrogen peroxide addition or glucose deprivation. Scaffolding protein functionality modulation Scaffolding proteins are large proteins often associated with GPCRs at the cell surface but also with cytoskeletal entities. These proteins assemble multiple signaling factors into coherent cascades of functional relevance. These scaffolding proteins are thought to be the basis of GPCR selectivity of function. The ability of these scaffolding proteins to still function in the face of disease-mimicking stressors (see above) has yet to the thoroughly investigated. We have been developing technologies to isolate and purify these scaffolding molecules both from clonal human neuronal cells and also from primary animal tissue from disease models. The two GPCR-interacting scaffolding proteins we have been investigating are the G protein coupled receptor kinase-interacting transcript (GIT) and spinophilin. The first protein has been demonstrated to link GPCR desensitization to the ability of the stimulated receptor to affect cytoskeletal dynamics. This will be of great importance at the functional synapse. We are currently generating stable SH-SY5Y cell lines expressing epitope tagged (FLAG and haemagglutinin) and GFP-tagged forms of these proteins for live confocal microscopy. In addition we have created our own affinity matrix purification systems to isolated specifically large amounts of GIT from cells and tissues through chemical combination of GIT antisera to an immobilized agarose phase. This process has also bee repeated for the second scaffolding protein, spinophilin. This protein, specifically enriched in the dendritic spines of neurons (of particular importance for control of synaptic transmission) also can act as a novel scaffolding protein than becomes recruited to the GPCR upon ligand activation. We are currently analyzing the functional signaling proteins that interact with these scaffolding factors in the presence or absence of disease mimetics. Receptor microdomain study The interactions that GPCRs make with other functional transmembrane proteins are primarily controlled via their association with microdomain regions of the plasma membrane. Such microdomain regions include focal adhesions, clathrin-coated pits and lipid rafts. We have begun studying how GPCR-interacting proteins, such as the beta-arrestins, can control the presence of GPCRs in these microdomains is affected by neurodegenerative disorders such as AD. To this end we are in the process of generating SH-SY5Y cell lines overexpressing various forms of the beta-arrestin molecules. With purification of both clathrin pits and lipid raft structures we are intending to identify how the presence of the GPCRs in various different plasma membrane compartments can affect their pharmacology, with respect to ligand interaction and also their downstream signaling ability. In addition to this we have been elucidating the nature of the connection between GPCR activity and the enzymatic function of the gamma-secretase complex that is specifically enriched in the same compartment as many GPCRs, e.g. the lipid raft. We have been employing both proteomic identification and in vitro enzymatic assays to demonstrate how acute receptor stimulation may control minute-to-minute beta amyloid production.

研究摘要我提出的研究涉及对三种不同水平受体介导的信号转导对照的年龄和疾病依赖性改变的研究。这三个控制级别是：受体辅助蛋白的信号转导调节；神经元细胞中的脚手架蛋白质功能；受体微域环境及其对受体功能的影响。我本人和我的研究研究员一直在进行以下研究以阐明这些领域。受体配件蛋白调节我们已经产生了两组稳定表达的人神经元细胞系（SH-SY5Y），表达了一个表现为表位标记的人M1和M2毒蕈碱乙酰胆碱G蛋白偶联受体（GPCR）。这些受体已被选择，因为它们代表了治疗阿尔茨海默氏病（AD）的最重要，最容易发生的治疗靶标之一，例如目前唯一处方的AD治疗方法AricePTTM是通过促进这些靶标的刺激而起作用的。我们选择了每种细胞系的24个克隆，并将其用于蛋白质组学研究。我们需要生成这些克隆的需求属于隔离大量受体蛋白所需的纯化技术，以便可以通过串联质谱法鉴定其相关的辅助蛋白。使用针对受体功能区域的血清的常规基于基于抗体的技术，可能会忽略受体同工型之间的细微超微结构变化。我们的假设是，在衰老和疾病期间，受体的化学计量与其辅助蛋白发生变化，然后这会影响受体与其同源配体的相互作用。因此，我们需要明确纯化跨膜受体至关重要。我们已经成功纯化了高数量的毒蕈碱受体，并证明了大量辅助蛋白可以与GPCR相关联。现在，我们打算将这些相关蛋白与疾病状态模型中与受体相互作用的蛋白质组进行比较，即以过氧化氢或葡萄糖剥夺的形式添加外源淀粉样蛋白β肽或氧化应激。脚手架蛋白功能调制脚手架蛋白是通常与细胞表面和细胞骨架实体相关的大蛋白。这些蛋白质将多个信号传导因子汇集到功能相关性相干的级联反应中。这些脚手架蛋白被认为是功能选择性的基础。这些脚手架蛋白在面对疾病的压力源时仍起作用的能力（见上文）尚未对其进行彻底研究。我们一直在开发技术来分离和纯化这些脚手架分子，既可以从克隆人的神经元细胞，也是从疾病模型中的原代动物组织中分离出来的。我们一直在研究的两个GPCR互动脚手架蛋白是G蛋白偶联受体激酶相互作用转录物（GIT）和脊柱蛋白酶。已经证明了第一种蛋白质将GPCR脱敏与刺激受体影响细胞骨架动力学的能力联系起来。在功能突触中，这将非常重要。目前，我们正在生成稳定的SH-SY5Y细胞系，表达标记的表位（FLAG和Haemagglutinin）和这些蛋白的GFP标记形式，用于实时共聚焦显微镜。此外，我们还创建了自己的亲和力矩阵纯化系统，通过将git抗血清到固定化琼脂糖相的化学组合从细胞和组织中分离出大量的git。该过程也重复了第二个脚手架蛋白自旋蛋白的蜜蜂。这种蛋白质（特异性地富含神经元的树突状刺（对于控制突触传播特别重要）也可以充当一种新型的脚手架蛋白，而不是在配体激活后被募集到GPCR上。我们目前正在分析在存在或不存在疾病模拟物的情况下与这些脚手架因子相互作用的功能信号蛋白。受体微域研究 GPCR与其他功能性跨膜蛋白的相互作用主要通过其与质膜的微区域的关联来控制。这样的微区区域包括局灶性粘附，涂有网状蛋白涂层的凹坑和脂质筏。我们已经开始研究GPCR相互作用的蛋白（例如β-arrestin）如何控制这些微区域中GPCR的存在受到神经退行性疾病（例如AD）的影响。为此，我们正在生成过表达β-arrestin分子的SH-SY5Y细胞系。通过纯化网格蛋白坑和脂质筏结构，我们打算确定在各种不同质膜室中GPCR的存在如何在配体相互作用以及其下游信号能力方面影响其药理。除此之外，我们一直在阐明GPCR活性与伽马分泌酶配合物的酶促功能之间的连接性质，这些伽马分泌酶配合物与许多GPCR相同，例如，例如脂质筏。我们一直在使用蛋白质组学鉴定和体外酶试验，以证明急性受体刺激如何控制微小的β-淀粉样蛋白产生。