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

 描述(申请人提供)：现在清楚的是，GPCR信号是多维的。确定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地针对药物发现的信号通路是至关重要的。受体的配体并不等同地激活所有的通路，而是会以牺牲其他通路为代价，表现出对某些通路的偏爱。信号通路特异性配体或有偏见的配体选择性地激活一条途径而不是另一条途径，在一条途径中发挥激动剂的作用，而在另一条途径中作为拮抗剂，极大地影响了我们对复杂的GPCR信号通路的理解和GPCR药物的发现。除了G蛋白和β-arrestin信号通路外，信号适配器蛋白14-3-3是GPCRs激活的另一种细胞效应物。α2-肾上腺素能受体(α2ARs)首次证实14-3-3是GPCRs的细胞效应因子。14-3-3与α2ARs的相互作用依赖于配体。14-3-3蛋白在细胞中普遍表达，但在大脑中表达最高。根据临床和实验室研究的证据，14-3-3蛋白与许多神经疾病有关，如阿尔茨海默病、帕金森氏病、精神分裂症和躁郁症。与β-arrestins类似，14-3-3蛋白没有内在的酶活性，但可以将两个或更多的蛋白质结合在一起，促进信号转导过程。最近的一项生物信息学分析预测，包括GRM在内的68%的神经递质GPCRs具有14-3-3个结合基序。有趣的是，GRM，即C家族GPCR，并不像许多GPCR在激活时那样招募β阻滞剂。除了G蛋白外，他们是否还利用14-3-3进行额外的信号传递？缺乏一个用户友好和可扩展的评估GPCR介导的14-3-3信号可能是信号通路未被利用的主要原因。我们计划应用LinkLight技术来开发GPCR介导的14-3-3信号通路分析。我们计划使用ADRB2作为模型来验证该检测的可行性。该方法利用ADRB2和14-3-3相互作用或形成ADRB2/14-3-3信号复合体作为信号读出。我们的初步数据(瞬时表达和稳定表达实验)表明，ADRB2/14-3-3相互作用或信号复合体的形成依赖于激动剂的浓度。我们还比较了ADRB2/14-3-3ɛ和ADRB2/β-arrestin-2LinkLight实验对不同激动剂、部分激动剂和拮抗剂的反应。初步数据显示，不同配基的总体反应模式相似，但在相对信号强度和效力(EC50)方面存在一些差异。有趣的是，ADRB2拮抗剂(基于G蛋白信号)在ADRB2/β-arrestin实验中显示部分活性，而在ADRB2/14-3-3实验中没有活性。我们的初步结果表明，根据不同的信号通路，配体并不是平等的。基于观察，它提示我们描述现有药物的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。它们在β-arrestin和14-3-3信号通路上的活性未知。尽管这些药物显示出了治疗效果，但也有严重的副作用。尽管制药业花费了巨大的努力，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特定的信号通路引起的吗？我计划使用D2R，一个重要的抗精神病药物靶点作为模型，来研究一组D2R配体对β的活性 Arrestin和14-3-3信号转导。现在已经知道，配体并不是平等地激活所有的途径，而是可以以牺牲其他途径为代价，表现出对某些途径的偏爱。除了G蛋白和β-arrestin信号外，配体还可能有14-3-3信号。有偏向的配体可能在这些信号通路上具有不同的活性。我们计划利用LinkLight技术的多路复用能力，建立一种同时评估β-arrestin和14-3-3信号的双信号通路分析方法。我们为提案计划了4项任务。Task1，以ADRB2为模型，评估GPCR介导的14-3-3信号转导，并建立稳定的14-3-3-pLuc报告细胞系。任务2，评估GPCR/14-3-3信号分析与脑源性GPCRs的普遍适用性。任务3，描述并比较D2R介导的14-3-3和β-arrestin信号通路与已知的D2R配体。任务4，建立GPCR14-3-3和GPCR14-3-3和GPCR14-β-arrestin双信号通路分析。该提案中开发的检测细胞系(工具)将可用于商业用途。我们还计划将这些工具用于商业化合物筛选和分析服务。一家CDA和MTA已经与一家美国主要研究试剂公司签署了潜在的商业机会合作协议。

 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.