Single-molecule imaging of GPCR-arrestin complexes

G蛋白偶联受体(GPCR)介导的信号转导是人类生理和生理功能的核心 疾病干预，但负责配体特异性信号反应的分子机制仍然存在 人们对此知之甚少。功能选择性或偏向激动论的发现已经从根本上影响了我们的 了解G蛋白偶联受体(GPCR)信号转导。偏向激动剂优先激活 特定的G蛋白依赖或非典型的G蛋白非依赖途径，包括由 Arrestin募集到受体上。除了在GPCR脱敏和贩运方面的作用外，逮捕措施 此外，支架信号通路不同于涉及G蛋白的信号通路。芳香素配基一直是 识别了许多不同的受体，包括多巴胺D2、血管紧张素和β-2(β2)肾上腺素能 感受器。例如，卡维地洛是一种芳香素偏向的β1和β2肾上腺素能受体(β2AR)激动剂 显示出有益的心脏保护作用。Arrestin特异性信号转导可能由激动剂诱导 受体内的构象变化和激动剂依赖的构象的形成 GPCR-arrestin复合体。最近的研究结果进一步表明，arrestin可能会保留“活性”构象 即使在脱离感受器之后。尽管arrestin特异性激动剂在gpr中起着关键作用。 在介导的信号转导中，我们缺乏对GPCR-arrestin复合体形成的分子和动力学的了解 有偏见的激动症的物质基础。这些信息对于合理设计药物是至关重要的 在特定效应器上的期望效果。集合光谱方法可以潜在地提供关键的见解 GPCR介导的信号传递，但这种大规模方法依赖于对平均反应的解释 从大量潜在的异质受体-效应器复合体中，掩盖关键信息 关于支撑功能的构象变化。因此，我们寻求建立一种手段来 直接量化单个GPCRs蛋白质在单分子尺度上的动态，使用广域，总 内反射荧光(TIRF)单分子荧光和荧光共振能 传输(FRET)成像方法。单分子成像提供了绕过以下限制的可能性 通过实现对随机的、异步构象的直接观测来进行系综测量 与功能关联的进程。使用这种方法，我们已经量化了G蛋白的配基效率 与β2肾上腺素能受体(β2AR)偶联。在拟议的措施中，我们将描述激动剂- 使用β2AR作为受体和arrestin复合体形成相关的依赖性构象变化 我们的模型系统。这些努力将首次揭示关键构象的顺序和时间 受体和arrestin两者的转变都是受体-arrestin相互作用所必需的，并与之相关， 对促进我们对arrestin激活机制和偏向本质的理解至关重要的信息 激动症对开发更具选择性的治疗药物具有重要的影响。

G protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-specific signaling responses remain poorly understood. The discovery of functional selectivity, or biased agonism, has fundamentally impacted our understanding of G protein-coupled receptor (GPCR) signaling. Biased agonists preferentially activate particular G protein-dependent or non-canonical G protein-independent pathways, including those mediated by arrestin recruitment to the receptor. In addition to their roles in GPCR desensitization and trafficking, arrestins also scaffold signaling pathways distinct from those involving G protein. Arrestin-biased ligands have been identified for a number of different receptors, including dopamine D2, angiotensin, and beta-2 (β2) adrenergic receptors. Carvedilol, for instance, is an arrestin-biased agonist for the β1 and β2 adrenergic receptors (β2AR) that exhibits beneficial cardioprotective effects. Arrestin-specific signaling is likely triggered by agonist-induced conformational changes within the receptor and the formation of agonist-dependent conformations of the GPCR-arrestin complex. Recent findings further suggest that arrestin may retain “active-like” conformations even after dissociating from the receptor. Although arrestin-specific agonism plays a critical role in GPCR- mediated signaling, we lack a molecular and kinetic understanding of how GPCR-arrestin complexes form and the physical basis of biased agonism. Such information is paramount to the rational design of drugs with desired efficacies at specific effectors. Ensemble spectroscopic methods can potentially provide critical insights into GPCR-mediated signaling, but bulk methods of this kind rely on the interpretation of average responses from very large numbers of potentially heterogeneous receptor-effector complexes, masking critical information about the conformational changes underpinning function. We have therefore sought to establish the means to directly quantify the dynamics of individual GPCRs proteins at the single-molecule scale using wide-field, total internal reflection fluorescence (TIRF) single-molecule fluorescence and fluorescence resonance energy transfer (FRET) imaging methods. Single-molecule imaging offers the potential to bypass the limitations of ensemble measurements by enabling direct observations of stochastic, asynchronous conformational processes associated with function. Using this approach, we have quantified ligand efficacy for G protein coupling to the beta2-adrenergic receptor (β2AR). In the proposed initiatives we will delineate the agonist- dependent conformational changes associated with receptor and arrestin complex formation using the β2AR as our model system. These efforts will reveal, for the first time, the order and timing of the key conformational transitions in both receptor and arrestin required for, and associated with, receptor-arrestin interaction, information vital to advancing our understanding of the arrestin activation mechanism and the nature of biased agonism with important ramifications for the development of more selective therapeutics.