Single molecule imaging of beta2 adrenergic receptor activation

描述(由申请人提供)：药理学理解的一个重要进展是功能选择性，即单个药物的不同下游效应物 G蛋白偶联受体(GPCR)可以被不同的配体差异激活。在一些不同的GPCR中已经发现了功能选择性的有趣例子，包括多巴胺受体和阿片受体，这是滥用药物的两个关键目标。尽管最近在GPCRs的结构生物学方面取得了巨大的进展，但药物结合调节受体活性的详细结构和动力学机制，特别是为什么不同的药物可以在同一受体上导致不同的作用，仍然知之甚少。我们提出了一系列关于β2肾上腺素能受体(B2AR)的单分子荧光共振能量转移(SmFRET)实验，以揭示其被不同配体差异激活的机制。SmFRET使跟踪单个分子随时间的移动成为可能，提供了一种直接测量功能过程中蛋白质构象瞬时变化的速率和幅度的方法。这类数据有望为阐明功能机制提供关键信息，包括检测静态和动态异质性以及瞬时填充、非累积的中间体，这些机制被系综方法掩盖。我们提出了以下具体目标：1)确定一系列配体对B2AR构象状态的平衡分布以及由smFRET确定的这些状态之间的跃迁速率的影响。2)探索杂三聚体G？S？？使用smFRET将复合体与激动剂激活的B2AR结合，并比较激动剂的光谱，以深入了解它们的信号差异。我们开发的技术平台将帮助我们了解不同B2AR激动剂的独特分子性质，并为设计具有优化性质的药物制定假设。此外，这些方法将广泛适用于其他GPCR的研究，这些GPCR是许多疾病的关键治疗靶点。

DESCRIPTION (provided by applicant): An important advance in pharmacological understanding is that of functional selectivity, whereby different downstream effectors of a single G protein-coupled receptor (GPCR) can be differentially activated by distinct ligands. Interesting examples of functional selectivity have been identified in a number of different GPCRs, including dopamine receptors and opiate receptors, two key targets of drugs of abuse. Despite enormous recent advances in the structural biology of GPCRs, the detailed structural and kinetic mechanisms by which drug binding modulates receptor activity, and particularly why different drugs can lead to different effects at the same receptor, remain poorly understood. We propose a series of single-molecule fluorescence resonance energy transfer (smFRET) experiments on the beta2 adrenergic receptor (B2AR) to delineate the mechanistic basis for its differential activation by different ligands. SmFRET makes it possible to follow the movements of an individual molecule over time, providing a means of making direct measurements of the rates and amplitudes of transient changes in protein conformation during function. Data of this kind hold the promise of providing critical information for elucidating functional mechanisms, including the detection of static and dynamic heterogeneities and transiently populated, non-accumulating intermediates, mechanisms that are masked by ensemble methods. We propose the following specific aims: 1) To determine the impact of a range of ligands on the equilibrium distribution of the conformational states of the B2AR and on the transition rates between these states, as determined by smFRET. 2) To probe structural dynamics within the heterotrimeric G?s?? complex bound to agonist-activated B2AR using smFRET and to compare a spectrum of agonists to provide insights into their signaling differences. The platform of technologies we develop will help us to understand the distinctive molecular properties of different B2AR agonists and to formulate hypotheses for designing drugs with optimized properties. Additionally, these methods will be broadly applicable to studies of other GPCRs that are critical therapeutic targets in numerous diseases.