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

项目摘要 除了有据可查的 G蛋白依赖型和 依赖于β-arrestin的gpr信号通路， 其他细胞效应器被招募到GPCRs。信号适配蛋白14-3-3就是这样的细胞效应器之一 与GPCRs接洽。14-3-3蛋白在细胞中广泛表达，但其表达水平最高 在大脑里。虽然生化证据表明14-3-3与一些GPCRs形成了络合物，但对14-3-3的研究 GPCR介导的14-3-3信号在很大程度上被忽视。缺乏评估特定14-3-3信号的能力 研究落后于G蛋白和β-arrestin信号通路研究的一个主要原因。 我们已经开发了一种新的方法来评估GPCR介导的14-3-3信号转导通过测量GPCR14-3-3信号 第一阶段研究中的3-3个相互作用。我们证明了GPCR介导的14-3-3信号是配体- 受监管的。多个GPCRs在激动剂刺激下与14-3-3蛋白相互作用。绿色聚合酶链式反应 14-3-3信号转导依赖于磷酸化，GPCR/14-3-3相互作用可能发生在受体之后 脱敏和内化。GPCR14-3-3信号转导可不依赖于β和Arrestin 激动剂可能在14-3-3和β-arrestin信号通路中具有不同的效力。GPCR还可以调解 14-3-3和Raf-1蛋白的相互作用。我们的工作开辟了一个新的广阔的领域，以前没有人欣赏过的gpcr。 信号转导。GPCR14-3-3LinkLight分析细胞为GPCR药物的发现提供了新的工具。 GPCR介导的14-3-3信号很可能是比我们以前更普遍的现象 意识到了。在第二阶段研究计划中，我们将继续表征和开发大量 商业GPCR/14-3-3细胞系检测脑源性GPCRs，包括5-羟色胺，多巴胺，阿片类药物， 食欲素、生长抑素、毒扁豆碱、大麻素、肾上腺素能和神经肽受体。这些受体- 介导的14-3-3信号通路尚未被单独描述。这些GPCR/14-3-3细胞系 这将是一种新的检测工具，有助于我们研究GPCR信号转导和开发新药。 我们还将研究代谢性谷氨酸受体(GPCR家族C成员)相互作用的可能性 含有14-3-3蛋白质。代谢性谷氨酸受体(GRM)可以通过G蛋白发出信号，但它们不能 募集β抑制蛋白，不存在β-arrestin信号。我们已经证明了GPCR介导的 以ADRB3为例，14-3-3信号通路可以是β-arrestin不依赖的(完成第一阶段 额外任务3)。因此，如果我们能证明代谢性谷氨酸受体可以介导14-3-3信号， 我们将有一种新的方法来瞄准这些重要的受体。 找到一个偏向的14-3-3信号配体将是另一个重要的发现。我们将与 洛克菲勒大学的托马斯·萨克马教授为试播屏幕。萨克马教授一直在研究 人多巴胺D4受体(HDRD4)在人类群体中有三个外显子变异 (D4.2、D4.4和D4.7)。我们将筛选在14-3-3和β-3之间具有差异信号的有偏见的配体 以D4.4变异体为模型研究ARRESIN信号转导途径。如果我们在屏幕上发现有偏见的点击，我们会 交叉核对不同变种的命中结果。有偏向的配体将是表征分子的有价值的探针 GPCR介导的14-3-3信号转导的生理意义

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.