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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要除了有据可查的 G蛋白依赖性和 β-抑制蛋白依赖性 GPCR 信号通路，其他细胞效应子被招募到 GPCR 中，信号接头蛋白 14-3-3 就是此类细胞效应子之一。 14-3-3 蛋白在细胞中普遍表达，但其表达量最高。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视。缺乏评估特定 14-3-3 信号传导的能力。研究落后于 G 蛋白和 β-arrestin 信号通路研究的一个主要原因。我们开发了一种新的检测方法，通过测量 GPCR 和 14-3-3 来评估 GPCR 介导的 14-3-3 信号传导。 1 期研究中的 3-3 相互作用证明 GPCR 介导的 14-3-3 信号传导是配体- 多个 GPCR 与 14-3-3 蛋白相互作用以响应 GPCR 介导的刺激。 14-3-3 信号传导依赖于磷酸化，GPCR/14-3-3 相互作用可能发生在受体之后 GPCR 介导的 14-3-3 信号传导可以是不依赖于 β-arrestin 的。激动剂在 14-3-3 中具有不同的效力，并且 β-arrestin 信号通路也可以介导。 14-3-3 和 Raf-1 激酶相互作用开辟了一个先前未被重视的 GPCR 的新领域。 GPCR/14-3-3 LinkLight 细胞为 GPCR 检测药物发现提供了新工具。 GPCR 介导的 14-3-3 信号传导可能是比我们之前发现的更普遍的现象在第二阶段研究计划中，我们将继续表征和开发大量的。用于脑源性 GPCR 的商业 GPCR/14-3-3 检测细胞系，包括血清素、多巴胺、阿片类药物、食欲素、生长抑素、毒蕈碱、大麻素、肾上腺素和神经肽受体。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。将成为帮助我们研究 GPCR 信号传导和开发新药的新型检测工具。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 14-3-3 蛋白的代谢性谷氨酸受体 (GRM) 可以通过 G 蛋白发出信号，但它们不这样做。招募 β-arrestin 并且没有 GPCR 介导的 β-arrestin 信号传导。以ADRB3为例，14-3-3信号通路可以是β-arrestin独立的（已完成Phase 1）额外任务 3) 因此，如果我们能够证明代谢谷氨酸受体可以介导 14-3-3 信号传导，我们将有一种新方法来针对这些重要受体。寻找有偏向的 14-3-3 信号配体将是我们将与之合作的另一个重要发现。洛克菲勒大学的 Thomas Sakmar 教授一直在研究该试验。人类多巴胺 D4 受体 (hDRD4)，在人群中存在三个外显子变异体（D4.2、D4.4 和 D4.7）我们将筛选在 14-3-3 和 β- 之间具有差异信号传导的偏向配体。如果我们在屏幕上发现有偏差的命中，我们将使用 D4.4 变体作为模型来观察抑制蛋白信号通路。交叉检查不同变体的命中结果将是表征配体的有价值的探针。 GPCR 介导的 14-3-3 信号传导的生理意义。