Mechanisms of drug action Advanced mass spectrometry methods enable monitoring of tens of thousands of phosphorylation sites in proteins. This technology can potentially distinguish cellular signaling pathways that produce beneficial effects from those that produce unwanted side effects. Liu et al. treated mice with various agonists of the kappa opioid receptor (a G protein–coupled receptor) and monitored changes in phosphorylation over time in different brain regions. The phosphorylation patterns revealed distinct patterns of signaling in various brain tissues, some of which were associated with unwanted side effects. Science, this issue p. eaao4927 High-throughput monitoring of phosphorylation helps define drug actions in the brain. INTRODUCTION The G protein–coupled receptor (GPCR) superfamily is a major drug target for neurological diseases. Functionally selective agonists activate GPCRs, such as the kappa opioid receptor (KOR), in a pathway-specific manner that may lead to drugs with fewer side effects. For example, KOR agonists that trigger beneficial antinociceptive, antipruritic, and anticonvulsant effects while causing minimal or no undesirable dysphoric, aversive, or psychotomimetic effects would be invaluable in light of the current opioid epidemic. However, functional selectivity observed in vitro frequently has little predictive value for behavioral outcomes. RATIONALE Obtaining a systems view of GPCR signaling in the brain may overcome the gap between in vitro pharmacology and in vivo testing. Recent breakthroughs in mass spectrometry–based proteomics have enabled us to quantify tens of thousands of phosphorylation events simultaneously in a high-throughput fashion. Using the KOR as a GPCR model, we applied this technology to achieve a global overview of the architecture of brain phosphoproteome in five mouse brain regions, in which we examined signaling induced by structurally and behaviorally diverse agonists. RESULTS Through the quantification of 50,000 different phosphosites, this approach yielded a brain region–specific systems view of the phosphoproteome, providing a context to understand KOR signaling in vivo. We observed strong regional specificity of KOR signaling attributable to differences in protein-protein interaction networks, neuronal contacts, and the different tissues in neuronal circuitries. Agonists with distinct signaling profiles elicited differential dynamic phosphorylation of synaptic proteins, thereby linking GPCR signaling to the modulation of brain functions. The most prominent changes occurred on synaptic proteins associated with dopaminergic, glutamatergic, and γ-aminobutyric acid–mediated (GABAergic) signaling and synaptic vesicle release. The large-scale dephosphorylation of synaptic proteins in the striatum after 5 min of agonist stimulation was partially blocked by protein phosphatase 2A (PP2A) inhibitors, underscoring the involvement of PP2A in KOR-mediated synaptic functions. Pathway analysis revealed enrichment of mTOR (mechanistic target of rapamycin) signaling by agonists associated with aversion. Strikingly, mTOR inhibition during KOR activation abolished aversion while preserving therapeutic antinociceptive and anticonvulsant effects. Parallel characterization of phosphoproteomic changes related to KOR-mediated mTOR activation in a cell line model provided additional mechanistic insights at the level of the signaling cascade. CONCLUSION We dissected the signaling pathways associated with desired and undesired outcomes of KOR activation in vivo and applied this knowledge to suppress the latter. Our work demonstrates the utility of combining phosphoproteomics with pharmacological tools and behavioral assessments as a general approach for studying GPCR signaling in vivo. Together with appropriate in vitro cellular systems, individual pathways can be characterized in depth, providing a rational basis for GPCR drug discovery. High-throughput phosphoproteomics to characterize in vivo brain GPCR signaling. Subsequent bioinformatic analysis enables prediction and modulation of downstream signaling pathways, which are correlated with unwanted effects but not the therapeutic outcome. A systems view of G protein–coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.

中文翻译：

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磷酸蛋白质组学阐明体内脑 GPCR 信号

药物作用机制 先进的质谱方法能够监测蛋白质中数以万计的磷酸化位点。这项技术可以潜在地将产生有益效果的细胞信号通路与产生不良副作用的细胞信号通路区分开来。刘等人。用各种κ阿片受体（一种G蛋白偶联受体）激动剂治疗小鼠，并监测不同大脑区域磷酸化随时间的变化。磷酸化模式揭示了各种脑组织中不同的信号模式，其中一些与不需要的副作用有关。科学，本期第 3 页。eaao4927 磷酸化的高通量监测有助于确定大脑中的药物作用。引言 G 蛋白偶联受体 (GPCR) 超家族是神经系统疾病的主要药物靶点。功能选择性激动剂以通路特异性方式激活 GPCR，例如 kappa 阿片受体 (KOR)，可能导致药物副作用更少。例如，鉴于当前的阿片类药物流行，KOR 激动剂会触发有益的镇痛、止痒和抗惊厥作用，同时引起最小的或没有不良的烦躁、厌恶或拟精神病作用，这将是无价的。然而，在体外观察到的功能选择性通常对行为结果几乎没有预测价值。基本原理 获得大脑中 GPCR 信号的系统视图可以克服体外药理学和体内测试之间的差距。基于质谱的蛋白质组学的最新突破使我们能够以高通量方式同时量化数以万计的磷酸化事件。使用 KOR 作为 GPCR 模型，我们应用该技术来实现对五个小鼠大脑区域中大脑磷酸蛋白质组结构的全局概览，其中我们检查了由结构和行为不同的激动剂诱导的信号传导。结果 通过对 50,000 个不同的磷酸化位点进行量化，这种方法产生了磷酸化蛋白质组的大脑区域特异性系统视图，为了解体内 KOR 信号传导提供了背景。我们观察到 KOR 信号的强烈区域特异性归因于蛋白质-蛋白质相互作用网络、神经元接触和神经元回路中不同组织的差异。具有不同信号分布的激动剂引起突触蛋白的差异动态磷酸化，从而将 GPCR 信号与大脑功能的调节联系起来。最显着的变化发生在与多巴胺能、谷氨酸能和γ-氨基丁酸介导的（GABA能）信号传导和突触小泡释放相关的突触蛋白上。激动剂刺激 5 分钟后纹状体中突触蛋白的大规模去磷酸化被蛋白磷酸酶 2A (PP2A) 抑制剂部分阻断，强调了 PP2A 参与 KOR 介导的突触功能。通路分析揭示了与厌恶相关的激动剂对 mTOR（雷帕霉素的机械靶点）信号传导的富集。引人注目的是，KOR 激活期间的 mTOR 抑制消除了厌恶，同时保留了治疗性镇痛和抗惊厥作用。在细胞系模型中对与 KOR 介导的 mTOR 激活相关的磷酸化蛋白质组学变化的平行表征提供了在信号级联水平上的额外机制见解。结论 我们剖析了与体内 KOR 激活的期望和不期望结果相关的信号通路，并应用这些知识来抑制后者。我们的工作证明了将磷酸蛋白质组学与药理学工具和行为评估相结合作为研究体内 GPCR 信号传导的一般方法的效用。与适当的体外细胞系统一起，可以深入表征各个途径，为 GPCR 药物发现提供合理的基础。用于表征体内脑 GPCR 信号的高通量磷酸蛋白质组学。随后的生物信息学分析能够预测和调节下游信号通路，这些信号通路与不良影响相关，但与治疗结果无关。G 蛋白偶联受体 (GPCR) 信号在其天然环境中的系统视图对于开发具有较少副作用的 GPCR 疗法至关重要。使用 kappa 阿片受体 (KOR) 作为模型，我们采用高通量磷酸蛋白质组学来研究由结构多样的激动剂在五个小鼠大脑区域中诱导的信号传导。50,000 种不同磷酸位点的量化提供了 KOR 体内信号传导的系统视图，揭示了药物作用的新机制。因此，我们发现了 U-50,488H 对雷帕霉素的机械靶点 (mTOR) 途径的富集，U-50,488H 是一种引起厌恶的激动剂，这是一种典型的 KOR 介导的副作用。因此，KOR 激活期间的 mTOR 抑制消除了厌恶，同时保留了有益的镇痛和抗惊厥作用。我们的结果确立了高通量磷酸蛋白质组学作为研究 GPCR 体内信号传导的一般策略，从而能够预测和调节行为结果。