Novel tools for investigating GPCR-mediated 14-3-3 signaling pathway

研究 GPCR 介导的 14-3-3 信号通路的新工具

• 批准号: 9345690
• 负责人: Haifeng Eishingdrelo
• 金额: $ 77.32万
• 依托单位: BIOINVENU CORPORATION
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9345690
• 关键词: ADRB2 gene; Adaptor Signaling Protein; Adrenergic Receptor; Adverse effects; Agonist; Alpha Cell; Anxiety; Arrestins; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Brain; CHRM3 gene; Cell Line; Cells; Collaborations; Collection; Complex; DRD2 gene; DRD4 gene; Development; Dopamine; Dopamine Receptor; Drug Receptors; Drug Targeting; Exons; Family; Firefly Luciferases; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Glutamate Receptor; Human; Individual; Investigation; Learning; Licensing; Ligand Binding; Ligands; Link; Luciferases; Maps; Measures; Mediating; Memory; Mental Health; Metabolic; Metabotropic Glutamate Receptors; Modeling; Mus; Muscarinic Acetylcholine Receptor; NIH Program Announcements; Neuropeptide Receptor; Neurotransmitter Receptor; Opioid; Pain; Perception; Pharmaceutical Preparations; Pharmacology; Phase; Phosphorylation; Physiological; Population; Proteins; Proto-Oncogene Proteins c-raf; Recruitment Activity; Reporter; Research; Research Personnel; Research Proposals; Respiration; Serotonin; Signal Pathway; Signal Transduction; Signaling Protein; Somatostatin Receptor; Technology; Time; Universities; Variant; Work; assay development; base; beta-arrestin; cannabinoid receptor; desensitization; drug development; drug discovery; hypocretin; member; novel; novel strategies; novel therapeutics; phase 1 study; professor; protein B; protein protein interaction; receptor; response; small molecule; tool; tool development

Project Summary In addition to well-documented G protein–dependent and β-arrestin-dependent GPCR signaling pathways, other cellular effectors are recruited to GPCRs. Signal adaptor protein 14-3-3 is one of such cellular effectors engaged with GPCRs. 14-3-3 proteins are ubiquitously expressed in cells, but their highest expression is found in the brain. Although biochemical evidence shows 14-3-3 forms complexes with some GPCRs, investigation of GPCR-mediated 14-3-3 signaling has been largely ignored. Lack of ability to assess specific 14-3-3 signaling is a major reason for studies to lag behind studies of G-protein and β-arrestin signaling pathways. We have developed a new assay for assessing GPCR-mediated 14-3-3 signaling by measuring GPCR and 14- 3-3 interactions in the phase 1 study. We demonstrate that GPCR-mediated 14-3-3 signaling is ligand- regulated. Multiple GPCRs interact with 14-3-3 proteins in response to agonist stimulation. GPCR-mediated 14-3-3 signaling is phosphorylation-dependent, and GPCR/14-3-3 interaction likely takes place after receptor desensitization and internalization. GPCR-mediated 14-3-3 signaling can be β-arrestin-independent and agonists can have different potencies in 14-3-3 and β-arrestin signaling pathways. GPCRs can also mediate 14-3-3 and Raf-1 kinase interaction. Our work opens up a new broad realm of previously unappreciated GPCR signal transduction. GPCR/14-3-3 LinkLight assay cells offer novel tools for GPCR drug discovery. It is likely that GPCR-mediated 14-3-3 signaling is a more general phenomenon than we have previously realized. In the Phase II research plan, we will continue characterizing and developing a large number of commercial GPCR/14-3-3 assay cell lines for brain-derived GPCRs including serotonin, dopamine, opioid, orexin, somatostatin, muscarinic, cannabinoid, adrenergic, and neuropeptide receptors. These receptors- mediated 14-3-3 signaling pathway has yet to be characterized individually. These GPCR/14-3-3 cell lines would be novel assay tools to aid us to study GPCR signaling and to develop new drugs. We will also investigate the potential that metabolic glutamate receptors (GPCR family C members) interact with 14-3-3 proteins. Metabolic glutamate receptors (GRMs) can signal through G-proteins, but they do not recruit β-arrestins and have no GPCR-mediated β-arrestin signaling. We have showed that GPCR-mediated 14-3-3 signaling pathway can be β-arrestin-independent by using ADRB3 as an example (completed Phase 1 extra task 3). Thus, if we can demonstrate that metabolic glutamate receptors can mediate 14-3-3 signaling, we will have a new approach to target these important receptors. Finding a biased 14-3-3 signaling ligand would be another important discovery. We will collaborate with Professor Thomas Sakmar in Rockefeller University for a pilot screen. Prof. Sakmar has been studying the human dopamine D4 receptor (hDRD4) for which there are three exon variants in the human populations (D4.2, D4.4 and D4.7). We will screen for biased ligands that have differential signaling between 14-3-3 and β- arrestin signaling pathways by using theD4.4 variant as the model. If we find biased hits in the screen, we will cross check the hits with different variants. Biased ligands would be valuable probes for characterizing the physiological significance of GPCR-mediated 14-3-3 signaling.

项目摘要 除了有据可查的 G蛋白依赖性和 β-arrestin依赖性GPCR信号通路， 其他细胞效应将募集到GPCR中。信号适配器蛋白14-3-3是这样的细胞效应之一 与GPCR互动。 14-3-3蛋白在细胞中普遍表达，但发现其最高表达 尽管生化证据表明14-3-3与某些GPCR形成复合物，但研究 GPCR介导的14-3-3信号传导在很大程度上被忽略了。缺乏评估特定14-3-3信号传导的能力是 研究落后于G蛋白和β-arrestin信号通路的主要原因。 我们已经开发了一种新的评估，用于通过测量GPCR和14-来评估GPCR介导的14-3-3信号传导。 1阶段研究中的3-3相互作用。我们证明了GPCR介导的14-3-3信号是配体 - 受监管。响应激动剂刺激，多个GPCR与14-3-3蛋白相互作用。 GPCR介导的 14-3-3信号转导依赖磷酸化，而GPCR/14-3-3的相互作用可能发生在接收器之后 脱敏和内在化。 GPCR介导的14-3-3信号传导可以是β-arrestin非依赖性的，并且 激动剂可以在14-3-3和β-arrestin信号通路中具有不同的电位。 GPCR也可以调解 14-3-3和RAF-1激酶相互作用。我们的工作打开了一个以前未欣赏的GPCR的新广泛领域 信号传输。 GPCR/14-3-3 Linklight Assay细胞提供了用于发现GPCR药物的新工具。 GPCR介导的14-3-3信号传导可能比以前更一般 实现。在第二阶段的研究计划中，我们将继续表征和开发大量 商业GPCR/14-3-3分析细胞系，用于脑衍生的GPCR，包括血清素，多巴胺，阿片类药物， Orexin，生长抑素，毒蕈碱，大麻素，肾上腺素和神经肽受体。这些受体 - 介导的14-3-3信号通路尚未单独表征。这些GPCR/14-3-3细胞系 将是帮助我们研究GPCR信号传导和开发新药的新型测定工具。 我们还将研究代谢谷氨酸受体（GPCR家族C成员）相互作用的潜力 具有14-3-3蛋白。代谢谷氨酸受体（GRM）可以通过G蛋白发出信号，但它们不会 募集β-arrestin，没有GPCR介导的β-arrestin信号传导。我们已经表明GPCR介导 14-3-3信号通路可以通过使用ADRB3作为示例（已完成的1阶段1 额外任务3）。如果我们可以证明代谢谷氨酸受体可以介导14-3-3信号传导，那 我们将有一种针对这些重要接收器的新方法。 找到偏见的14-3-3信号配体是另一个重要发现。我们将与 洛克菲勒大学的托马斯·萨克马教授为试点屏幕。萨克马教授一直在研究 人类多巴胺D4受体（HDRD4）在人群中有三种外显子变体 （D4.2，D4.4和D4.7）。我们将筛选具有14-3-3和β-之间具有差异信号的偏置配体。 通过使用THED4.4变体作为模型，阻止蛋白信号通路。如果我们在屏幕上发现有偏见的命中，我们将 用不同的变体交叉检查命中。有偏见的配体对于表征 GPCR介导的14-3-3信号传导的生理意义。