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

 描述（由申请人提供）：现在清楚的是，GPCR信号传导是多维的。定义受体信号通路的机制对于理解细胞生物学的许多方面以及有效靶向药物发现的信号通路至关重要。受体的配体不能同等地激活所有通路，而是可以表现出以牺牲其他通路为代价的对某些通路的偏好。信号通路特异性配体或偏向性配体选择性地激活一种通路而不是另一种通路，以及在一种通路中作为激动剂而在另一种通路中作为拮抗剂，这极大地影响了我们对复杂的GPCR信号通路和GPCR药物发现的理解。除了G蛋白和β-抑制蛋白信号通路之外，信号衔接蛋白14-3-3是另一种由GPCR激活的细胞效应物。14-3-3作为GPCR的细胞效应物的第一个证据是用α2-肾上腺素能受体（α 2AR）证明的。14-3-3与α 2 AR的相互作用是配体依赖性的。14-3-3蛋白在细胞中普遍表达，但在大脑中表达最高。基于来自临床和实验室研究的证据，14-3-3蛋白与许多神经系统疾病如阿尔茨海默病、帕金森病、精神分裂症、双相情感障碍有关。与β-抑制蛋白类似，14-3-3蛋白没有内在的酶活性，但将两种或多种蛋白结合在一起以促进信号转导过程。最近的生物信息学分析预测68%的神经递质GPCR包括GRM具有14-3-3结合基序。有趣的是，GRM，家族C GPCR，在激活时不像许多GPCR那样招募β-抑制蛋白。除了G蛋白外，它们还利用14-3-3进行额外的信号传导吗？缺乏用于评估GPCR介导的14-3-3信号传导的用户友好和可扩展的工具可能是信号传导途径未被开发的主要原因。我们计划应用LinkLight技术开发GPCR介导的14-3-3信号通路检测方法。我们计划使用ADRB 2作为模型来证明测定的可行性。该测定利用ADRB 2和14-3-3相互作用或ADRB 2/14-3-3信号复合物形成作为信号读数。我们的初步数据（瞬时表达和稳定表达实验）显示ADRB 2/14-3-3相互作用或信号复合物的形成是激动剂浓度依赖性的。我们还比较了ADRB 2/14-3-3抑制蛋白和ADRB 2/β-arrestin-2 LinkLight试验对各种激动剂、部分激动剂和拮抗剂的反应。初步数据显示，各种配体反应的总体模式相似，但相对信号强度和效力（EC 50）存在一些差异。有趣的是，ADRB 2拮抗剂（基于G蛋白信号传导）在ADRB 2/β-arrestin试验中显示部分活性，但在ADRB 2/14-3-3试验中无活性。我们的初步研究结果表明，配体是不平等的基础上不同的信号通路。基于这一观察，它促使我们分析现有药物的β-arrestin和14-3-3信号通路。从历史上看，GPCR药物的发现依赖于G蛋白信号通路来评估化合物的活性，例如许多旧的抗精神病药物。它们对β-抑制蛋白和14-3-3信号传导的活性是未知的。虽然已经证明了治疗效果，但这些药物也有严重的副作用。尽管制药业付出了巨大的努力，但开发更安全，更有效的抗精神病药物的选择仍然难以捉摸。治疗和副作用可能是由于特定的信号通路吗？我计划使用D2 R，一个重要的抗精神病药物靶点作为模型来研究一组D2 R配体对β-受体的活性， 抑制蛋白和14-3-3信号。现在已知的是，配体不能同等地激活所有途径，而是可以以牺牲其他途径为代价对某些途径表现出偏好。除了G蛋白和β-抑制蛋白信号传导外，配体还可以具有14-3-3信号传导。偏置配体可能对这些信号通路具有不同的活性。我们计划利用LinkLight技术的多重能力，建立同时评估β-arrestin和14-3-3信号传导的双信号传导途径测定。我们为提案制定了四项任务。任务1，使用ADRB 2作为模型来评估GPCR介导的14-3-3信号传导并产生稳定的14-3-3-pLuc报告细胞系。任务2，评估GPCR/14-3-3信号传导试验对脑源性GPCR的普遍适用性。任务3，分析并比较D2 R介导的14-3-3和β-arrestin信号通路与已知的D2 R配体。任务4，开发GPCR/14-3-3和GPCR/β-arrestin双信号通路测定。本提案中开发的试验细胞系（工具）将可商购获得。我们还计划将这些工具用于商业化合物筛选和分析服务。A CDA和MTA已与美国一家主要的研究试剂公司签署了潜在的商业合作机会。

 DESCRIPTION (provided by applicant): It is now clear that GPCR signaling is pluridimensional. Defining mechanisms for receptor signaling pathways is essential for understanding many aspects of cell biology, as well as for effectively targeting signaling pathways for drug discovery. Ligands of receptors do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others. Signaling pathway-specific ligands or biased ligands that selectively activate one pathway over another, as well as behave as agonists in one pathway but antagonists in another pathway have greatly impacted our understanding of intricate GPCR signaling pathways and GPCR drug discovery. In addition to G-protein and β-arrestin signaling pathways, signal adaptor protein 14-3-3 is another cellular effectors activated by GPCRs. The first evidence of 14-3-3 as a cellular effector of GPCRs was demonstrated with the α2-adrenergic receptors (α2ARs). Interaction of 14-3-3 and α2ARs is ligand-dependent. 14-3-3 proteins are ubiquitously expressed in cells, but their highest expression is found in the brain. 14-3-3 proteins have been implicated in a number of neurological disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, bipolar disorder based on evidence from both clinical and laboratory studies. Similar to β-arrestins,14-3-3 proteins have no intrinsic enzymatic activity, but bring two or more proteins together to facilitate signal transduction processes. A very recent bioinformatic analysis predicts 68% neurotransmitter GPCRs including GRMs have 14-3-3 binding motifs. Interestingly, GRMs, family C GPCRs, do not recruit β-arrestins like many GPCRs do when activated. Do they utilize 14-3-3 for additional signaling in addition to G-proteins? Lack of a user-friendly and scalable too for assessing GPCR-mediated 14-3-3 signaling could be a major reason for the signaling pathway unexploited. We plan to apply LinkLight technology to develop GPCR mediated 14-3-3 signaling pathway assays. We plan to use ADRB2 as a model to demonstrate the assay feasibility. The assay utilizes ADRB2 and 14-3-3 interaction or ADRB2/14-3-3 signal complex formation as the signal readout. Our preliminary data (transient expression and stable expression experiments) showed ADRB2/14-3-3 interaction or signal complex formation is agonist concentration-dependent. We also compared ADRB2/14-3-3ɛ and ADRB2/β-arrestin-2 LinkLIght assays in response to various agonists, partial agonists, and antagonists. The preliminary data showed that the overall patterns of various ligand responses were similar, but there were some differences in relative signal strength and potency (EC50). Interestingly, ADRB2 antagonists (based on G-protein signaling) showed a partial activity in ADRB2/β-arrestin assay, but had no activity in ADRB2/14-3-3 assay. Our preliminary results showed that ligands are not created equal based on different signaling pathways. Based on the observation, it prompts us to profile existing drugs for their β-arrestin and 14-3-3 signaling pathways. Historically, GPCR drug discoveries rely on G-protein signaling pathways to assess compound activity such as many old antipsychotic drugs. Their activity on β-arrestin and 14-3-3 signaling i unknown. Although having demonstrated therapeutic benefits, these drugs also have serious side effects. Despite huge efforts spent by the pharmaceutical industry, the options for developing safer and more efficacious antipsychotic drugs remain elusive. Could therapeutic and side effects are due to specific signaling pathways? I plan to use D2R, an important antipsychotic drug target as a model to investigate a panel of D2R ligands for their activity on β arrestin and 14-3-3 signaling. It is now known that ligands do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others. Ligands could have 14-3-3 signaling in addition to G-protein and β-arrestin signaling. Biased ligands could have differential activities on these signaling pathways. We plan to take the advantage of multiplex ability of the LinkLight technology to establish a dual-signaling pathway assay for assessing β-arrestin and 14-3-3 signaling simultaneously. We plan 4 tasks for the proposal. Task1, Using ADRB2 as a model to assess GPCR-mediated 14-3-3 signaling and generating stable 14-3-3-pLuc reporter cell lines. Task 2, Assess the general applicability of GPCR/14-3-3 signaling assays with brain-derived GPCRs. Task 3, Profile and compare D2R-mediated 14-3-3 and β-arrestin signaling pathways with known D2R ligands. Task 4, Develop GPCR/14-3-3 and GPCR/β-arrestin dual- signaling pathway assays. The assay cell lines (tools) developed in the proposal will be commercially available. We also plan to use the tools for commercial compound screening and profiling services. A CDA and MTA have signed with a major US research reagent company for potential business opportunity collaboration.