G PROTEIN SUBUNIT FUNCTION

G 蛋白亚基功能

基本信息

批准号：
7368294
负责人：
NARASIMHAN GAUTAM
金额：
$ 31.78万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-03-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7368294
关键词：
Acylation Affinity Agonist Binding Cell membrane Cells Characteristics Complex Disease Endoplasmic Reticulum Eukaryota Eukaryotic Cell Family G-Protein-Coupled Receptors GTP-Binding Proteins Goals Golgi Apparatus Heterotrimeric GTP-Binding Proteins Image Imaging Techniques Intracellular Membranes Life Localized Mammalian Cell Measurement Measures Mediating Membrane Protein Traffic Methods Modeling Movement Numbers Pharmaceutical Preparations Process Protein Subunits Proteins Receptor Activation Reporting Role Role playing therapy Screening procedure Signal Pathway Signal Transduction Site Surface Tertiary Protein Structure Testing Therapeutic Thinking Today base deacylation extracellular follow-up intracellular protein transport member novel novel therapeutics protein function protein localization location receptor response sensor therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Heterotrimeric G proteins (123) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Observation of the spatio-temporal dynamics of G protein localization in living mammalian cells using various imaging techniques showed unexpectedly that G protein subunits shuttle rapidly (t1/2 <1 min) between the plasma membrane and intracellular membranes. Consistent with such shuttling, G protein pools are constitutively maintained in endomembranes and receptor activation results in six out of the twelve members of the G protein 3 subunit family translocating to endomembranes as 23 complexes. The long term goal is to determine if shuttling and receptor mediated translocation are widespread among various eukaryotes and are central processes in signaling that have remained undiscovered. The specific aims are (i) to identify mechanisms at the basis of G protein shuttling, 23 translocation and differential targeting to intracellular membranes. (ii) To test the hypothesis that receptor induced 23 translocation controls the magnitude of amplification of downstream signaling. (iii) To identify the role of the translocated 23 complex in endomembranes. Sensitive methods that measure receptor-G protein interaction will be used to test the model that affinity of 3 subunit types for receptors regulates translocation. The finding that shuttling and translocation are acylation dependent and a palmitoylated PDZ domain protein inhibits translocation will be followed up to identify potential binding partners that mediate shuttling through an acylation/deacylation cycle. Live cell measurements of G protein effector activity will be used to examine the role of receptor induced 23 complex translocation on downstream activity at the plasma membrane. The possibility that translocation of 23 complexes to endomembranes regulates Ras activity and membrane trafficking will be tested using sensors that report these activities in live cells. The unexpected finding that G proteins continually shuttle between the PM and endomembranes facilitating receptor mediated translocation of a large number of 23 subunit types indicates the presence of novel mechanisms and functions that have not been anticipated. Elucidating these mechanisms and functions can provide newer therapeutic sites and targets for diseases caused by aberrant signaling. More than 30% of the commercially available therapeutic drugs today are targeted at G protein coupled receptors. The rapid reversible translocation of fluorescent protein tagged 3 subunits in response to agonists and antagonists provides a tool for non-invasively and rapidly performing high throughput and high content screening for potential therapeutic molecules that act on these receptors.G proteins mediate most signaling pathways in cells and their receptors are one of the most important targets of therapeutic drugs. Unanticipated movement of G proteins between the external and internal membranes of cells has been discovered. Identifying the mechanistic and functional bases of this movement can provide newer therapeutic sites and targets for diseases caused by aberrant signaling.

描述（由申请人提供）：异三聚体G蛋白（123）介导哺乳动物细胞中的大多数信号通路。长期以来，G蛋白功能位于质膜。使用各种成像技术观察到活哺乳动物细胞中G蛋白定位的时空动力学观察，出乎意料地表明，G蛋白亚基在质膜和细胞内膜之间迅速（T1/2 <1分钟）迅速穿梭（T1/2 <1分钟）。与此类穿梭一致，G蛋白池在内膜和受体激活中的组成型维持，导致G蛋白3亚基家族的十二个成员中有六个转移到子宫内膜为23个复合物。长期目标是确定各种真核生物之间的穿梭和受体介导的易位是否普遍存在，并且是信号传导中未被发现的中心过程。具体目的是（i）基于G蛋白穿梭，23易位和对细胞内膜的差异靶向的机制。（ii）检验以下假设：受体诱导23易位控制下游信号传导的扩增大小。（iii）确定易位23个复合物在内膜中的作用。测量受体-G蛋白相互作用的敏感方法将用于测试3种亚基类型对受体的亲和力调节易位的模型。将跟踪穿梭和易位是酰化依赖性和棕榈酰化的PDZ结构蛋白抑制易位的发现，以识别潜在的结合伴侣，这些伴侣通过酰化/脱酰化循环介导穿梭的伴侣。 G蛋白效应活性的活细胞测量将用于检查受体诱导的23个复合物在质膜下游活性上的作用。使用在活细胞中报告这些活性的传感器测试了23个复合物向内膜的易位调节RAS活性和膜运输的可能性。出乎意料的发现，G蛋白在PM和内膜之间不断穿梭，促进受体介导的大量23种亚基类型的易位表明存在尚未预期的新机制和功能。阐明这些机制和功能可以为异常信号引起的疾病提供更新的治疗部位和靶标。如今，超过30％的市售治疗药物针对G蛋白偶联受体。荧光蛋白标记的3个亚基的快速可逆转运为激动剂和拮抗剂提供了一种工具，可为对这些受体作用的潜在治疗分子进行非侵入性和快速执行的高吞吐量和高含量筛选。G蛋白介导细胞及其受体中最重要的靶标的最重要的靶标是最重要的药物。已经发现了G蛋白在细胞外部和内部膜之间的意外运动。识别该运动的机械和功能基础可以为异常信号引起的疾病提供更新的治疗部位和靶标。